U.S. patent application number 14/131984 was filed with the patent office on 2014-10-02 for high-performance ketol-acid reductoisomerases.
This patent application is currently assigned to CALIFORNIA INSTITUTE OF TECHNOLOGY. The applicant listed for this patent is Sabine Bastian, Doug Lies, Peter Meinhold, Stephanie Porter-Scheinman, Sebastian Schoof, Christopher Smith, Christopher Snow. Invention is credited to Sabine Bastian, Doug Lies, Peter Meinhold, Stephanie Porter-Scheinman, Sebastian Schoof, Christopher Smith, Christopher Snow.
Application Number | 20140295513 14/131984 |
Document ID | / |
Family ID | 51621225 |
Filed Date | 2014-10-02 |
United States Patent
Application |
20140295513 |
Kind Code |
A1 |
Meinhold; Peter ; et
al. |
October 2, 2014 |
High-Performance Ketol-Acid Reductoisomerases
Abstract
The present invention relates to recombinant microorganisms
comprising at least one nucleic acid molecule encoding a ketol-acid
reductoisomerase (KARI) or a modified NADH-dependent variant
thereof, wherein said KARI is at least about 80% identical to SEQ
ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,
SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58. The present
invention also relates to recombinant microorganisms comprising at
least one nucleic acid molecule encoding a ketol-acid
reductoisomerase (KARI) or a modified NADH-dependent variant
thereof, wherein said KARI is at least about 99% identical to SEQ
ID NO: 64. In various aspects of the invention, the recombinant
microorganisms may comprise an isobutanol producing metabolic
pathway and can be used in methods of making isobutanol.
Inventors: |
Meinhold; Peter; (Topanga,
CA) ; Lies; Doug; (Englewood, CO) ;
Porter-Scheinman; Stephanie; (Conifer, CO) ; Smith;
Christopher; (Parker, CO) ; Snow; Christopher;
(Englewood, CO) ; Bastian; Sabine; (Pasadena,
CA) ; Schoof; Sebastian; (Pasadena, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Meinhold; Peter
Lies; Doug
Porter-Scheinman; Stephanie
Smith; Christopher
Snow; Christopher
Bastian; Sabine
Schoof; Sebastian |
Topanga
Englewood
Conifer
Parker
Englewood
Pasadena
Pasadena |
CA
CO
CO
CO
CO
CA
CA |
US
US
US
US
US
US
US |
|
|
Assignee: |
CALIFORNIA INSTITUTE OF
TECHNOLOGY
Pasadena
CA
GEVO, INC.
Englewood
CO
|
Family ID: |
51621225 |
Appl. No.: |
14/131984 |
Filed: |
July 11, 2012 |
PCT Filed: |
July 11, 2012 |
PCT NO: |
PCT/US12/46185 |
371 Date: |
June 19, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13303884 |
Nov 23, 2011 |
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14131984 |
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61506562 |
Jul 11, 2011 |
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61506564 |
Jul 11, 2011 |
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61510618 |
Jul 22, 2011 |
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Current U.S.
Class: |
435/160 ;
435/190; 435/254.2; 536/23.1 |
Current CPC
Class: |
C12Y 101/01086 20130101;
Y02E 50/10 20130101; C12P 7/16 20130101; C12N 9/0006 20130101 |
Class at
Publication: |
435/160 ;
435/190; 435/254.2; 536/23.1 |
International
Class: |
C12P 7/16 20060101
C12P007/16; C12N 9/04 20060101 C12N009/04 |
Goverment Interests
ACKNOWLEDGMENT OF GOVERNMENTAL SUPPORT
[0002] This invention was made with government support under
Contract No. 2009-10006-05919, awarded by the United States
Department of Agriculture, and under Contract No. W911NF-09-2-0022,
awarded by the United States Army Research Laboratory. The
government has certain rights in the invention.
Claims
1.-72. (canceled)
73. A mutant ketol-acid reductoisomerase (KARI) comprising one or
more mutations or modifications at positions corresponding to amino
acids selected from the group consisting of: (a) valine 48 of the
L. lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis
KARI (SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID
NO: 10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e)
glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f);
leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89
of the L. lactis KARI (SEQ ID NO: 10); (h) lysine 118 of the L.
lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L. lactis
KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis
KARI (SEQ ID NO: 10).
74. The mutant KARI of claim 73, wherein said valine 48 is replaced
with a residue selected from leucine or proline.
75. The mutant KARI of claim 73, wherein said arginine 49 is
replaced with a residue selected from valine, leucine, serine, or
proline.
76. The mutant KARI of claim 73, wherein said lysine 52 is replaced
with a residue selected from leucine, alanine, isoleucine,
methionine, phenylalanine, tryptophan, tyrosine, valine, aspartic
acid, or glutamic acid.
77. The mutant KARI of claim 73, wherein said serine 53 is replaced
with a residue selected from aspartic acid or glutamic acid.
78. The mutant KARI of claim 73, wherein said glutamic acid 59 is
replaced with a residue selected from lysine, arginine, or
histidine.
79. The mutant KARI of claim 73, wherein said leucine 85 is
replaced with a residue selected from threonine or alanine.
80. The mutant KARI of claim 73, wherein said isoleucine 89 is
replaced with alanine.
81. The mutant KARI of claim 73, wherein said lysine 118 is
replaced with a residue selected from glutamic acid or aspartic
acid.
82. The mutant KARI of claim 73, wherein said threonine 182 is
replaced with a residue selected from serine, asparagine, or
glutamine.
83. The mutant KARI of claim 73, wherein said glutamic acid 320 is
replaced with a residue selected from lysine, arginine, or
histidine.
84. The mutant KARI of claim 73, wherein said mutant KARI is a
modified version of a wild-type KARI, and wherein said wild-type
KARI is at least 80% identical to SEQ ID NO: 10.
85.-93. (canceled)
94. A recombinant microorganism comprising at least one nucleic
acid molecule encoding a mutant KARI of claim 73.
95. (canceled)
96. An isolated nucleic acid molecule encoding a mutant KARI,
wherein said mutant KARI comprises one or more mutations or
modifications at positions corresponding to amino acids selected
from the group consisting of: (a) valine 48 of the L. lactis KARI
(SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI (SEQ ID NO:
10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO: 10); (d)
serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e) glutamic acid
59 of the L. lactis KARI (SEQ ID NO: 10): (f); leucine 85 of the L.
lactis KARI (SEQ ID NO: 10); (g) isoleucine 89 of the L. lactis
KARI (SEQ ID NO: 10); (h) lysine 118 of the L. lactis KARI (SEQ ID
NO: 10); (i) threonine 182 of the L. lactis KARI (SEQ ID NO: 10);
and (j) glutamic acid 320 of the L. lactis KARI (SEQ ID NO:
10).
97.-98. (canceled)
99. The recombinant microorganism of claim 94, wherein said
recombinant microorganism further expresses exogenous genes
encoding an acetolactate synthase, a dihydroxy acid dehydratase, a
keto-isovalerate decarboxylase, and an alcohol dehydrogenase,
wherein said recombinant microorganism produces isobutanol.
100. (canceled)
101. The recombinant microorganism of claim 94, wherein said
recombinant microorganism is a yeast microorganism.
102. A method of producing isobutanol, comprising: (a) providing a
recombinant microorganism of claim 99; and (b) cultivating the
recombinant microorganism in a culture medium containing a
feedstock providing a carbon source, until the isobutanol is
produced.
103. A method of producing isobutanol, comprising: (a) providing a
recombinant microorganism of claim 101; and (b) cultivating the
recombinant microorganism in a culture medium containing a
feedstock providing a carbon source, until the isobutanol is
produced.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/506,562, filed Jul. 11, 2011, U.S.
Provisional Application Ser. No. 61/506,564, filed Jul. 11, 2011,
U.S. Provisional Application Ser. No. 61/510,618, filed Jul. 22,
2011, and is a continuation-in-part of U.S. Non-Provisional
application Ser. No. 13/303,884, filed Nov. 23, 2011, each of which
is herein incorporated by reference in its entirety for all
purposes.
TECHNICAL FIELD
[0003] Recombinant microorganisms and methods of producing such
microorganisms are provided. Also provided are methods of producing
beneficial metabolites including fuels and chemicals by contacting
a suitable substrate with the recombinant microorganisms of the
invention and enzymatic preparations therefrom.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0004] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
GEVO.sub.--064.sub.--01WO_SeqList_ST25.txt, date recorded: Jul. 10,
2012, file size: 185 kilobytes).
BACKGROUND
[0005] The ability of microorganisms to convert sugars to
beneficial metabolites including fuels, chemicals, and amino acids
has been widely described in the literature in recent years. See,
e.g., Alper et al., 2009, Nature Microbiol. Rev. 7: 715-723 and
McCourt et al., 2006, Amino Acids 31: 173-210. Recombinant
engineering techniques have enabled the creation of microorganisms
that express biosynthetic pathways capable of producing a number of
useful products, including the commodity chemical, isobutanol.
[0006] Isobutanol, also a promising biofuel candidate, has been
produced in recombinant microorganisms expressing a heterologous,
five-step metabolic pathway (See, e.g., WO/2007/050671 to Donaldson
et al., WO/2008/098227 to Liao et al., and WO/2009/103533 to Festel
et al.). However, the microorganisms produced to date have fallen
short of commercial relevance due to their low performance
characteristics, including, for example low productivities, low
titers, and low yields.
[0007] The second step of the isobutanol producing metabolic
pathway is catalyzed by ketol-acid reductoisomerase (KARI), which
converts acetolactate to 2,3-dihydroxyisovalerate. Because KARI is
an essential enzyme in the isobutanol production pathway, it is
desirable that recombinant microorganisms engineered to produce
isobutanol exhibit optimal KARI activity. The present application
addresses this need by identifying several KARI enzymes that give
high performance with an isobutanol production pathway expressed in
yeast. Accordingly, this application describes methods of
increasing isobutanol production through the use of recombinant
microorganisms comprising KARI enzymes with improved properties for
the production of isobutanol.
[0008] Another important feature of a KARI enzyme is the ability to
use NADH as a cofactor for the conversion of acetolactate to
2,3-dihydroxyisovalerate. The present inventors have found that
when an NADH-dependent KARI is used in conjunction with an
NADH-dependent alcohol dehydrogenase (ADH), isobutanol can be
produced at theoretical yield and/or under anaerobic conditions.
See, e.g., commonly owned and co-pending US Publication No. US
2010/0143997. Because NADH-dependence is an important feature of a
KARI enzyme, the present inventors have identified several
beneficial mutations which can be made to the KARI enzymes
identified herein to switch the cofactor specificity of the enzymes
from NADPH to NADH.
SUMMARY OF THE INVENTION
[0009] The present inventors have discovered a group of KARI
enzymes with high level activity in the isobutanol pathway. The use
of one or more of these KARI enzymes, or NADH-dependent variants
thereof, can improve production of the isobutanol.
[0010] In a first aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 80% identical to SEQ ID NO: 2. In one embodiment,
the KARI is derived from the genus Shewanella. In a specific
embodiment, the KARI is derived from Shewanella sp. strain MR-4. In
another specific embodiment, the KARI is encoded by SEQ ID NO:
1.
[0011] In another aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 80% identical to SEQ ID NO: 4. In one embodiment,
the KARI is derived from the genus Vibrio. In a specific
embodiment, the KARI is derived from Vibrio fischeri. In another
specific embodiment, the KARI is encoded by SEQ ID NO: 3.
[0012] In another aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 80% identical to SEQ ID NO: 6. In one embodiment,
the KARI is derived from the genus Gramella. In a specific
embodiment, the KARI is derived from Gramella forsetii. In another
specific embodiment, the KARI is encoded by SEQ ID NO: 5.
[0013] In another aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 80% identical to SEQ ID NO: 8. In one embodiment,
the KARI is derived from the genus Cytophaga. In a specific
embodiment, the KARI is derived from Cytophaga hutchinsonii. In
another specific embodiment, the KARI is encoded by SEQ ID NO:
7.
[0014] In another aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 80% identical to SEQ ID NO: 10. In one embodiment,
the KARI is derived from a genus selected from Lactococcus and
Streptococcus. In a specific embodiment, the KARI is derived from
Lactococcus lactis, Streptococcus equinus, or Streptococcus
infantarius. In another specific embodiment, the KARI is encoded by
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID
NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO:
25.
[0015] In another aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 80% identical to SEQ ID NO: 28. In one embodiment,
the KARI is derived from the genus Methanococcus. In a specific
embodiment, the KARI is derived from Methanococcus maripaludis,
Methanococcus vannielii, or Methanococcus voltae. In another
specific embodiment, the KARI is encoded by SEQ ID NO: 27, SEQ ID
NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO:
37.
[0016] In another aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 80% identical to SEQ ID NO: 40. In one embodiment,
the KARI is derived from a genus selected from Zymomonas,
Erythrobacter, Sphingomonas, Sphingobium, and Novosphingobium. In a
specific embodiment, the KARI is derived from Zymomonas mobilis,
Erythrobacter litoralis, Sphingomonas wittichii, Sphingobium
japonicum, Sphingobium chlorophenolicum, or Novosphingobium
nitrogenifigens. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:
45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO:
53.
[0017] In another aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 80% identical to SEQ ID NO: 56. In one embodiment,
the KARI is derived from the genus Bacteroides. In a specific
embodiment, the KARI is derived from Bacteroides thetaiotaomicron.
In another specific embodiment, the KARI is encoded by SEQ ID NO:
55.
[0018] In another aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 80% identical to SEQ ID NO: 58. In one embodiment,
the KARI is derived from the genus Schizosaccharomyces. In a
specific embodiment, the KARI is derived from Schizosaccharomyces
pombe or Schizosaccharomyces japonicus. In another specific
embodiment, the KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or
SEQ ID NO: 61.
[0019] In another aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 99% identical to SEQ ID NO: 64. In one embodiment,
the KARI is derived from the genus Salmonella. In a specific
embodiment, the KARI is derived from Salmonella enterica. In
another specific embodiment, the KARI is encoded by SEQ ID NO:
63.
[0020] In some embodiments, the KARI may be modified to be
NADH-dependent. Accordingly, the present application further
relates to NADH-dependent ketol-acid reductoisomerases (NKRs)
derived from a KARI that is at least about 80% identical to SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ
ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58. Thus, in one
embodiment, the present application relates to a recombinant
microorganism comprising a NKR derived from a KARI that is at least
about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ
ID NO: 58.
[0021] In other embodiments, the present application further
relates to NADH-dependent ketol-acid reductoisomerases (NKRs)
derived from a KARI that is at least about 99% identical to SEQ ID
NO: 64. Thus, in one embodiment, the present application relates to
a recombinant microorganism comprising a NKR derived from a KARI
that is at least about 99% identical to SEQ ID NO: 64.
[0022] Therefore, the present application also relates to mutated
ketol-acid reductoisomerase (KARI) enzymes that utilize NADH rather
than NADPH.
[0023] Examples of such KARIs include enzymes having one or more
modifications or mutations at positions corresponding to amino
acids selected from: (a) alanine 71 of the Shewanella sp. KARI (SEQ
ID NO: 2); (b) arginine 76 of the Shewanella sp. KARI (SEQ ID NO:
2); (c) serine 78 of the Shewanella sp. KARI; and (d) glutamine 110
of the Shewanella sp. KARI (SEQ ID NO: 2).
[0024] In one embodiment, the KARI enzyme contains a modification
or mutation at the amino acid corresponding to position 71 of the
Shewanella sp. KARI (SEQ ID NO: 2). In another embodiment, the KARI
enzyme contains a modification or mutation at the amino acid
corresponding to position 76 of the Shewanella sp. KARI (SEQ ID NO:
2). In yet another embodiment, the KARI enzyme contains a
modification or mutation at the amino acid corresponding to
position 78 of the Shewanella sp. KARI (SEQ ID NO: 2). In yet
another embodiment, the KARI enzyme contains a modification or
mutation at the amino acid corresponding to position 110 of the
Shewanella sp. KARI (SEQ ID NO: 2). In one embodiment, the KARI
enzyme contains two or more modifications or mutations at the amino
acids corresponding to the positions described above. In another
embodiment, the KARI enzyme contains three or more modifications or
mutations at the amino acids corresponding to the positions
described above. In yet another embodiment, the KARI enzyme
contains four modifications or mutations at the amino acids
corresponding to the positions described above. Further included
within the scope of the application are KARI enzymes, other than
the Shewanella sp. KARI (SEQ ID NO: 2), which contain modifications
or mutations corresponding to those set out above. In an exemplary
embodiment, the modified KARI is derived from a KARI that is at
least about 80% identical to SEQ ID NO: 2.
[0025] Additional mutated ketol-acid reductoisomerase (KARI)
enzymes that utilize NADH rather than NADPH include enzymes having
one or more modifications or mutations at positions corresponding
to amino acids selected from: (a) valine 48 of the L. lactis KARI
(SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI (SEQ ID NO:
10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO: 10); (d)
serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e) glutamic acid
59 of the L. lactis KARI (SEQ ID NO: 10): (f); leucine 85 of the L.
lactis KARI (SEQ ID NO: 10); (g) isoleucine 89 of the L. lactis
KARI (SEQ ID NO: 10); (h) lysine 118 of the L. lactis KARI (SEQ ID
NO: 10); (i) threonine 182 of the L. lactis KARI (SEQ ID NO: 10);
and (j) glutamic acid 320 of the L. lactis KARI (SEQ ID NO:
10).
[0026] In one embodiment, the KARI enzyme contains a modification
or mutation at the amino acid corresponding to position 48 of the
L. lactis KARI (SEQ ID NO: 10). In another embodiment the KARI
enzyme contains a modification or mutation at the amino acid
corresponding to position 49 of the L. lactis KARI (SEQ ID NO: 10).
In yet another embodiment, the KARI enzyme contains a modification
or mutation at the amino acid corresponding to position 52 of the
L. lactis KARI (SEQ ID NO: 10). In yet another embodiment, the KARI
enzyme contains a modification or mutation at the amino acid
corresponding to position 53 of the L. lactis KARI (SEQ ID NO: 10).
In yet another embodiment, the KARI enzyme contains a modification
or mutation at the amino acid corresponding to position 59 of the
L. lactis KARI (SEQ ID NO: 10). In yet another embodiment, the KARI
enzyme contains a modification or mutation at the amino acid
corresponding to position 85 of the L. lactis KARI (SEQ ID NO: 10).
In yet another embodiment, the KARI enzyme contains a modification
or mutation at the amino acid corresponding to position 89 of the
L. lactis KARI (SEQ ID NO: 10). In yet another embodiment, the KARI
enzyme contains a modification or mutation at the amino acid
corresponding to position 118 of the L. lactis KARI (SEQ ID NO:
10). In yet another embodiment, the KARI enzyme contains a
modification or mutation at the amino acid corresponding to
position 182 of the L. lactis KARI (SEQ ID NO: 10). In yet another
embodiment, the KARI enzyme contains a modification or mutation at
the amino acid corresponding to position 320 of the L. lactis KARI
(SEQ ID NO: 10). In one embodiment, the KARI enzyme contains two or
more modifications or mutations at the amino acids corresponding to
the positions described above. In another embodiment, the KARI
enzyme contains three or more modifications or mutations at the
amino acids corresponding to the positions described above. In yet
another embodiment, the KARI enzyme contains four or more
modifications or mutations at the amino acids corresponding to the
positions described above. In yet another embodiment, the KARI
enzyme contains five or more modifications or mutations at the
amino acids corresponding to the positions described above. In yet
another embodiment, the KARI enzyme contains six or more
modifications or mutations at the amino acids corresponding to the
positions described above. In yet another embodiment, the KARI
enzyme contains seven or more modifications or mutations at the
amino acids corresponding to the positions described above. In yet
another embodiment, the KARI enzyme contains eight or more
modifications or mutations at the amino acids corresponding to the
positions described above. In yet another embodiment, the KARI
enzyme contains nine or more modifications or mutations at the
amino acids corresponding to the positions described above. In yet
another embodiment, the KARI enzyme contains ten modifications or
mutations at the amino acids corresponding to the positions
described above. Further included within the scope of the
application are KARI enzymes, other than the L. lactis KARI (SEQ ID
NO: 10), which contain modifications or mutations corresponding to
those set out above. In an exemplary embodiment, the modified KARI
is derived from a KARI that is at least about 80% identical to SEQ
ID NO: 10.
[0027] Additional mutated ketol-acid reductoisomerase (KARI)
enzymes that utilize NADH rather than NADPH include enzymes having
one or more modifications or mutations at positions corresponding
to amino acids selected from: (a) alanine 71 of the S. enterica
KARI (SEQ ID NO: 64); (b) arginine 76 of the S. enterica KARI (SEQ
ID NO: 64); (c) serine 78 of the S. enterica KARI (SEQ ID NO: 64);
(d) glutamine 110 of the S. enterica KARI (SEQ ID NO: 64); (e)
aspartic acid 146 of the S. enterica KARI (SEQ ID NO: 64); (f)
glycine 185 of the S. enterica KARI (SEQ ID NO: 64); and (g) lysine
433 of the S. enterica KARI (SEQ ID NO: 64).
[0028] In one embodiment, the KARI enzyme contains a modification
or mutation at the amino acid corresponding to position 71 of the
S. enterica KARI (SEQ ID NO: 64). In another embodiment, the KARI
enzyme contains a modification or mutation at the amino acid
corresponding to position 76 of the S. enterica KARI (SEQ ID NO:
64). In yet another embodiment, the KARI enzyme contains a
modification or mutation at the amino acid corresponding to
position 78 of the S. enterica KARI (SEQ ID NO: 64). In yet another
embodiment, the KARI enzyme contains a modification or mutation at
the amino acid corresponding to position 110 of the S. enterica
KARI (SEQ ID NO: 64). In yet another embodiment, the KARI enzyme
contains a modification or mutation at the amino acid corresponding
to position 146 of the S. enterica KARI (SEQ ID NO: 64). In yet
another embodiment, the KARI enzyme contains a modification or
mutation at the amino acid corresponding to position 185 of the S.
enterica KARI (SEQ ID NO: 64). In yet another embodiment, the KARI
enzyme contains a modification or mutation at the amino acid
corresponding to position 433 of the S. enterica KARI (SEQ ID NO:
64). In one embodiment, the KARI enzyme contains two or more
modifications or mutations at the amino acids corresponding to the
positions described above. In another embodiment, the KARI enzyme
contains three or more modifications or mutations at the amino
acids corresponding to the positions described above. In yet
another embodiment, the KARI enzyme contains four or more
modifications or mutations at the amino acids corresponding to the
positions described above. In yet another embodiment, the KARI
enzyme contains five or more modifications or mutations at the
amino acids corresponding to the positions described above. In yet
another embodiment, the KARI enzyme contains six or more
modifications or mutations at the amino acids corresponding to the
positions described above. In yet another embodiment, the KARI
enzyme contains seven modifications or mutations at the amino acids
corresponding to the positions described above. Further included
within the scope of the application are KARI enzymes, other than
the S. enterica KARI (SEQ ID NO: 64), which contain modifications
or mutations corresponding to those set out above. In an exemplary
embodiment, the modified KARI is derived from a KARI that is at
least about 99% identical to SEQ ID NO: 64.
[0029] In various embodiments described herein, the modified or
mutated KARI may exhibit an increased catalytic efficiency with
NADH as compared to the wild-type KARI. In one embodiment, the KARI
has at least about a 5% increased catalytic efficiency with NADH as
compared to the wild-type KARI. In another embodiment, the KARI has
at least about a 25%, at least about a 50%, at least about a 75%,
at least about a 100%, at least about a 500%, at least about 1000%,
or at least about a 10000% increased catalytic efficiency with NADH
as compared to the wild-type KARI.
[0030] In various embodiments described herein, the modified or
mutated KARI may exhibit a decreased Michaelis Menten constant
(K.sub.M) for NADH as compared to the wild-type KARI. In one
embodiment, the KARI has at least about a 5% decreased K.sub.M for
NADH as compared to the wild-type KARI. In another embodiment, the
KARI has at least about a 25%, at least about a 50%, at least about
a 75%, at least about a 90%, at least about a 95%, or at least
about a 97.5% decreased K.sub.M for NADH as compared to the
wild-type KARI.
[0031] In various embodiments described herein, the modified or
mutated KARI may exhibit an increased catalytic constant
(k.sub.cat) with NADH as compared to the wild-type KARI. In one
embodiment, the KARI has at least about a 5% increased k.sub.cat
with NADH as compared to the wild-type KARI. In another embodiment,
the KARI has at least about a 25%, at least about a 50%, at least
about a 75%, at least about 100%, at least about 200%, or at least
about a 500% increased k.sub.cat with NADH as compared to the
wild-type KARI.
[0032] In various embodiments described herein, the modified or
mutated KARI may exhibit an increased Michaelis Menten constant
(K.sub.M) for NADPH as compared to the wild-type KARI. In one
embodiment, the KARI has at least about a 5% increased K.sub.M for
NADPH as compared to the wild-type KARI. In another embodiment, the
KARI has at least about a 25%, at least about a 50%, at least about
a 100%, at least about a 500%, at least about a 1000%, or at least
about a 5000% increased K.sub.M for NADPH as compared to the
wild-type KARI.
[0033] In various embodiments described herein, the modified or
mutated KARI may exhibit a decreased catalytic constant (k.sub.cat)
with NADPH as compared to the wild-type KARI. In one embodiment,
the KARI has at least about a 5% decreased k.sub.cat with NADPH as
compared to the wild-type KARI. In another embodiment, the KARI has
at least about a 25%, at least about a 50%, or at least about a
75%, at least about 90% decreased k.sub.cat with NADPH as compared
to the wild-type KARI.
[0034] In some embodiments described herein, the catalytic
efficiency of the modified or mutated KARI with NADH is increased
with respect to the catalytic efficiency with NADPH of the
wild-type KARI. In one embodiment, the catalytic efficiency of said
KARI with NADH is at least about 10% of the catalytic efficiency
with NADPH of the wild-type KARI. In another embodiment, the
catalytic efficiency of said KARI with NADH is at least about 25%,
at least about 50%, or at least about 75% of the catalytic
efficiency with NADPH of the wild-type KARI. In some embodiments,
the modified or mutated KARI preferentially utilizes NADH rather
than NADPH.
[0035] In one embodiment, the application is directed to
NADH-dependent KARI enzymes having a catalytic efficiency with NADH
that is greater than the catalytic efficiency with NADPH. In one
embodiment, the catalytic efficiency of the NADH-dependent KARI is
at least about 2-fold greater with NADH than with NADPH. In another
embodiment, the catalytic efficiency of the NADH-dependent KARI is
at least about 4-fold, at least about 5-fold, at least about
6-fold, at least about 7-fold, at least about 8-fold, at least
about 9-fold, at least about 10-fold, at least about 25-fold, at
least about 50-fold, at least about 100-fold, or at least about
500-fold greater with NADH than with NADPH.
[0036] In one embodiment, the application is directed to modified
or mutated KARI enzymes that demonstrate a switch in cofactor
specificity from NADPH to NADH. In one embodiment, the modified or
mutated KARI has at least about a 2:1 ratio of catalytic efficiency
(k.sub.cat/K.sub.M) with NADH over k.sub.cat with NADPH. In an
exemplary embodiment, the modified or mutated KARI has at least
about a 10:1 ratio of catalytic efficiency (k.sub.cat/K.sub.M) with
NADH over catalytic efficiency (k.sub.cat/K.sub.M) with NADPH.
[0037] In one embodiment, the KARI exhibits at least about a 1:10
ratio of K.sub.M for NADH over K.sub.M for NADPH.
[0038] In additional embodiments, the application is directed to
modified or mutated KARI enzymes that have been codon optimized for
expression in certain desirable host organisms, such as yeast and
E. coli.
[0039] In another aspect, the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a KARI enzyme. In one embodiment, the nucleic acid
molecule encodes a KARI that is at least about 80% identical to SEQ
ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,
SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58. In another
embodiment, the nucleic acid molecule encodes a KARI this is at
least about 99% identical to SEQ ID NO: 64. In yet another
embodiment, the nucleic acid molecule encodes an NADH-dependent
ketol-acid reductoisomerase (NKR) derived from a KARI that is at
least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or
SEQ ID NO: 58. In one embodiment, the nucleic acid molecule encodes
an NADH-dependent ketol-acid reductoisomerase (NKR) derived from a
KARI that is at least about 80% identical to SEQ ID NO: 2, wherein
said NKR has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) alanine 71 of the
Shewanella sp. KARI (SEQ ID NO: 2); (b) arginine 76 of the
Shewanella sp. KARI (SEQ ID NO: 2): (c) serine 78 of the Shewanella
sp. KARI; and (d) glutamine 110 of the Shewanella sp. KARI (SEQ ID
NO: 2). In another embodiment, the nucleic acid molecule encodes an
NADH-dependent ketol-acid reductoisomerase (NKR) derived from a
KARI that is at least about 80% identical to SEQ ID NO: 10, wherein
said NKR has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) valine 48 of the L.
lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI
(SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO:
10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e)
glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f);
leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89
of the L. lactis KARI (SEQ ID NO: 10); (h) lysine 118 of the L.
lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L. lactis
KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis
KARI (SEQ ID NO: 10). In one embodiment, the nucleic acid molecule
encodes an NADH-dependent ketol-acid reductoisomerase (NKR) derived
from a KARI that is at least about 99% identical to SEQ ID NO: 64.
In a further embodiment, the nucleic acid molecule encodes an
NADH-dependent ketol-acid reductoisomerase (NKR) derived from a
KARI that is at least about 99% identical to SEQ ID NO: 64, wherein
said NKR has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) alanine 71 of the
S. enterica KARI (SEQ ID NO: 64); (b) arginine 76 of the S.
enterica KARI (SEQ ID NO: 64); (c) serine 78 of the S. enterica
KARI (SEQ ID NO: 64); (d) glutamine 110 of the S. enterica KARI
(SEQ ID NO: 64); (e) aspartic acid 146 of the S. enterica KARI (SEQ
ID NO: 64); (f) glycine 185 of the S. enterica KARI (SEQ ID NO:
64); and (g) lysine 433 of the S. enterica KARI (SEQ ID NO:
64).
[0040] In various embodiments described in the application, the
recombinant microorganism comprises an isobutanol producing
metabolic pathway. In one embodiment, the isobutanol producing
metabolic pathway comprises at least one exogenous gene encoding a
polypeptide that catalyzes a step in the conversion of pyruvate to
isobutanol. In another embodiment, the isobutanol producing
metabolic pathway comprises at least two exogenous genes encoding
polypeptides that catalyze steps in the conversion of pyruvate to
isobutanol. In yet another embodiment, the isobutanol producing
metabolic pathway comprises at least three exogenous genes encoding
polypeptides that catalyze steps in the conversion of pyruvate to
isobutanol. In yet another embodiment, the isobutanol producing
metabolic pathway comprises at least four exogenous genes encoding
polypeptides that catalyze steps in the conversion of pyruvate to
isobutanol. In yet another embodiment, the isobutanol producing
metabolic pathway comprises at least five exogenous genes encoding
polypeptides that catalyze steps in the conversion of pyruvate to
isobutanol. In yet another embodiment, all of the isobutanol
producing metabolic pathway steps in the conversion of pyruvate to
isobutanol are converted by exogenously encoded enzymes. In an
exemplary embodiment, at least one of the exogenously encoded
enzymes is a KARI that is at least about 80% identical to SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ
ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58. In another exemplary
embodiment, at least one of the exogenously encoded enzymes is a
KARI that is at least about 99% identical to SEQ ID NO: 64. In yet
another exemplary embodiment, at least one of the exogenously
encoded enzymes is a KARI enzyme has one or more modifications or
mutations at positions corresponding to amino acids selected from:
(a) alanine 71 of the Shewanella sp. KARI (SEQ ID NO: 2); (b)
arginine 76 of the Shewanella sp. KARI (SEQ ID NO: 2); (c) serine
78 of the Shewanella sp. KARI; and (d) glutamine 110 of the
Shewanella sp. KARI (SEQ ID NO: 2). In yet another exemplary
embodiment, at least one of the exogenously encoded enzymes is a
KARI enzyme has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) valine 48 of the L.
lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI
(SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO:
10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e)
glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f);
leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89
of the L. lactis KARI (SEQ ID NO: 10); (h) lysine 118 of the L.
lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L. lactis
KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis
KARI (SEQ ID NO: 10). In yet another exemplary embodiment, at least
one of the exogenously encoded enzymes is a KARI enzyme has one or
more modifications or mutations at positions corresponding to amino
acids selected from: (a) alanine 71 of the S. enterica KARI (SEQ ID
NO: 64); (b) arginine 76 of the S. enterica KARI (SEQ ID NO: 64);
(c) serine 78 of the S. enterica KARI (SEQ ID NO: 64): (d)
glutamine 110 of the S. enterica KARI (SEQ ID NO: 64); (e) aspartic
acid 146 of the S. enterica KARI (SEQ ID NO: 64); (f) glycine 185
of the S. enterica KARI (SEQ ID NO: 64); and (g) lysine 433 of the
S. enterica KARI (SEQ ID NO: 64).
[0041] In one embodiment, one or more of the isobutanol pathway
genes encodes an enzyme that is localized to the cytosol. In one
embodiment, the recombinant microorganisms comprise an isobutanol
producing metabolic pathway with at least one isobutanol pathway
enzyme localized in the cytosol. In another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least two isobutanol pathway enzymes
localized in the cytosol. In yet another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least three isobutanol pathway enzymes
localized in the cytosol. In yet another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least four isobutanol pathway enzymes
localized in the cytosol. In an exemplary embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with five isobutanol pathway enzymes localized in
the cytosol. In yet another exemplary embodiment, the recombinant
microorganisms comprise an isobutanol producing metabolic pathway
with all isobutanol pathway enzymes localized in the cytosol.
[0042] In various embodiments described herein, the isobutanol
pathway genes may encode enzyme(s) selected from the group
consisting of acetolactate synthase (ALS), ketol-acid
reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD),
2-keto-acid decarboxylase, e.g., keto-isovalerate decarboxylase
(KIVD), and alcohol dehydrogenase (ADH). In one embodiment, the
KARI is an NADH-dependent KARI (NKR). In another embodiment, the
ADH is an NADH-dependent ADH. In yet another embodiment, the KARI
is an NADH-dependent KARI (NKR) and the ADH is an NADH-dependent
ADH. In an exemplary embodiment, the KARI is at least about 80%
identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58.
In another exemplary embodiment, the KARI is at least about 99%
identical to SEQ ID NO: 64. In yet another exemplary embodiment,
the KARI comprises one or more modifications or mutations at
positions corresponding to amino acids selected from: (a) alanine
71 of the Shewanella sp. KARI (SEQ ID NO: 2); (b) arginine 76 of
the Shewanella sp. KARI (SEQ ID NO: 2); (c) serine 78 of the
Shewanella sp. KARI; and (d) glutamine 110 of the Shewanella sp.
KARI (SEQ ID NO: 2). In yet another exemplary embodiment, the KARI
comprises one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) valine 48 of the L.
lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI
(SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO:
10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e)
glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f);
leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89
of the L. lactis KARI (SEQ ID NO: 10); (h) lysine 118 of the L.
lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L. lactis
KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis
KARI (SEQ ID NO: 10). In yet another exemplary embodiment, the KARI
comprises one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) alanine 71 of the
S. enterica KARI (SEQ ID NO: 64); (b) arginine 76 of the S.
enterica KARI (SEQ ID NO: 64); (c) serine 78 of the S. enterica
KARI (SEQ ID NO: 64): (d) glutamine 110 of the S. enterica KARI
(SEQ ID NO: 64); (e) aspartic acid 146 of the S. enterica KARI (SEQ
ID NO: 64); (f) glycine 185 of the S. enterica KARI (SEQ ID NO:
64); and (g) lysine 433 of the S. enterica KARI (SEQ ID NO:
64).
[0043] In various embodiments described herein, the recombinant
microorganisms of the invention that comprise an isobutanol
producing metabolic pathway may be further engineered to reduce or
eliminate the expression or activity of one or more enzymes
selected from a pyruvate decarboxylase (PDC), a
glycerol-3-phosphate dehydrogenase (GPD), a 3-keto acid reductase
(3-KAR), or an aldehyde dehydrogenase (ALDH).
[0044] In various embodiments described herein, the recombinant
microorganisms may be recombinant yeast microorganisms. In some
embodiments, the recombinant yeast microorganisms may be members of
the Saccharomyces clade, Saccharomyces sensu stricto
microorganisms, Crabtree-negative yeast microorganisms,
Crabtree-positive yeast microorganisms, post-WGD (whole genome
duplication) yeast microorganisms, pre-WGD (whole genome
duplication) yeast microorganisms, and non-fermenting yeast
microorganisms.
[0045] In some embodiments, the recombinant microorganisms may be
yeast recombinant microorganisms of the Saccharomyces clade.
[0046] In some embodiments, the recombinant microorganisms may be
Saccharomyces sensu stricto microorganisms. In one embodiment, the
Saccharomyces sensu stricto is selected from the group consisting
of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S.
uvarum, S. carocanis and hybrids thereof.
[0047] In some embodiments, the recombinant microorganisms may be
Crabtree-negative recombinant yeast microorganisms. In one
embodiment, the Crabtree-negative yeast microorganism is classified
into a genera selected from the group consisting of Saccharomyces,
Kluyveromyces, Pichia, Issatchenkia, Hansenula, or Candida. In
additional embodiments, the Crabtree-negative yeast microorganism
is selected from Saccharomyces kluyveri, Kluyveromyces lactis,
Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Hansenula
anomala, Candida utilis and Kluyveromyces waltii.
[0048] In some embodiments, the recombinant microorganisms may be
Crabtree-positive recombinant yeast microorganisms. In one
embodiment, the Crabtree-positive yeast microorganism is classified
into a genera selected from the group consisting of Saccharomyces,
Kluyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia and
Schizosaccharomyces. In additional embodiments, the
Crabtree-positive yeast microorganism is selected from the group
consisting of Saccharomyces cerevisiae, Saccharomyces uvarum,
Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces
castelli, Kluyveromyces thermotolerans, Candida glabrata, Z.
bailli, Z. rouxii, Debaryomyces hansenii, Pichia pastorius,
Schizosaccharomyces pombe, and Saccharomyces uvarum.
[0049] In some embodiments, the recombinant microorganisms may be
post-WGD (whole genome duplication) yeast recombinant
microorganisms. In one embodiment, the post-WGD yeast recombinant
microorganism is classified into a genera selected from the group
consisting of Saccharomyces or Candida. In additional embodiments,
the post-WGD yeast is selected from the group consisting of
Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces
bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and
Candida glabrata.
[0050] In some embodiments, the recombinant microorganisms may be
pre-WGD (whole genome duplication) yeast recombinant
microorganisms. In one embodiment, the pre-WGD yeast recombinant
microorganism is classified into a genera selected from the group
consisting of Saccharomyces, Kluyveromyces, Candida, Pichia,
Issatchenkia, Debaryomyces, Hansenula, Pachysolen, Yarrowia and
Schizosaccharomyces. In additional embodiments, the pre-WGD yeast
is selected from the group consisting of Saccharomyces kluyveri,
Kluyveromyces thermotolerans, Kluyveromyces marxianus,
Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicalis,
Pichia pastoris, Pichia anomala, Pichia stipitis, Issatchenkia
orientalis, Issatchenkia occidentalis, Debaryomyces hansenii,
Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, and
Schizosaccharomyces pombe.
[0051] In some embodiments, the recombinant microorganisms may be
microorganisms that are non-fermenting yeast microorganisms,
including, but not limited to those, classified into a genera
selected from the group consisting of Tricosporon, Rhodotorula,
Myxozyma, or Candida. In a specific embodiment, the non-fermenting
yeast is C. xestobii.
[0052] In another aspect, the present invention provides methods of
producing isobutanol using a recombinant microorganism as described
herein. In one embodiment, the method includes cultivating the
recombinant microorganism in a culture medium containing a
feedstock providing the carbon source until a recoverable quantity
of isobutanol is produced and optionally, recovering the
isobutanol. In one embodiment, the microorganism produces
isobutanol from a carbon source at a yield of at least about 5
percent theoretical. In another embodiment, the microorganism
produces isobutanol at a yield of at least about 10 percent, at
least about 15 percent, about least about 20 percent, at least
about 25 percent, at least about 30 percent, at least about 35
percent, at least about 40 percent, at least about 45 percent, at
least about 50 percent, at least about 55 percent, at least about
60 percent, at least about 65 percent, at least about 70 percent,
at least about 75 percent, at least about 80 percent, at least
about 85 percent, at least about 90 percent, at least about 95
percent, or at least about 97.5 percent theoretical.
[0053] In one embodiment, the recombinant microorganism converts
the carbon source to isobutanol under aerobic conditions. In
another embodiment, the recombinant microorganism converts the
carbon source to isobutanol under microaerobic conditions. In yet
another embodiment, the recombinant microorganism converts the
carbon source to isobutanol under anaerobic conditions.
BRIEF DESCRIPTION OF DRAWINGS
[0054] Illustrative embodiments of the invention are illustrated in
the drawings, in which:
[0055] FIG. 1 illustrates an exemplary embodiment of an isobutanol
pathway.
[0056] FIG. 2 illustrates an exemplary embodiment of an
NADH-dependent isobutanol pathway.
DETAILED DESCRIPTION
[0057] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a polynucleotide" includes a plurality of such polynucleotides and
reference to "the microorganism" includes reference to one or more
microorganisms, and so forth.
[0058] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods, devices and
materials are described herein.
[0059] Any publications discussed above and throughout the text are
provided solely for their disclosure prior to the filing date of
the present application. Nothing herein is to be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior disclosure.
[0060] The term "microorganism" includes prokaryotic and eukaryotic
microbial species from the Domains Archaea, Bacteria and Eucarya,
the latter including yeast and filamentous fungi, protozoa, algae,
or higher Protista. The terms "microbial cells" and "microbes" are
used interchangeably with the term microorganism.
[0061] The term "prokaryotes" is art recognized and refers to cells
which contain no nucleus or other cell organelles. The prokaryotes
are generally classified in one of two domains, the Bacteria and
the Archaea. The definitive difference between organisms of the
Archaea and Bacteria domains is based on fundamental differences in
the nucleotide base sequence in the 16S ribosomal RNA.
[0062] The term "Archaea" refers to a categorization of organisms
of the division Mendosicutes, typically found in unusual
environments and distinguished from the rest of the prokaryotes by
several criteria, including the number of ribosomal proteins and
the lack of muramic acid in cell walls. On the basis of ssrRNA
analysis, the Archaea consist of two phylogenetically-distinct
groups: Crenarchaeota and Euryarchaeota. On the basis of their
physiology, the Archaea can be organized into three types:
methanogens (prokaryotes that produce methane); extreme halophiles
(prokaryotes that live at very high concentrations of salt (NaCl);
and extreme (hyper) thermophiles (prokaryotes that live at very
high temperatures). Besides the unifying archaeal features that
distinguish them from Bacteria (i.e., no murein in cell wall,
ester-linked membrane lipids, etc.), these prokaryotes exhibit
unique structural or biochemical attributes which adapt them to
their particular habitats. The Crenarchaeota consist mainly of
hyperthermophilic sulfur-dependent prokaryotes and the
Euryarchaeota contain the methanogens and extreme halophiles.
[0063] "Bacteria", or "eubacteria", refers to a domain of
prokaryotic organisms. Bacteria include at least eleven distinct
groups as follows: (1) Gram-positive (gram+) bacteria, of which
there are two major subdivisions: (1) high G+C group
(Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C
group (Bacillus, Clostridia, Lactobacillus, Staphylococci,
Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple
photosynthetic+non-photosynthetic Gram-negative bacteria (includes
most "common" Gram-negative bacteria); (3) Cyanobacteria, e.g.,
oxygenic phototrophs; (4) Spirochetes and related species; (5)
Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8)
Green sulfur bacteria; (9) Green non-sulfur bacteria (also
anaerobic phototrophs); (10) Radioresistant micrococci and
relatives; (11) Thermotoga and Thermosipho thermophiles.
[0064] "Gram-negative bacteria" include cocci, nonenteric rods, and
enteric rods. The genera of Gram-negative bacteria include, for
example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia,
Francisella, Haemophilus, Bordetella, Escherichia, Salmonella,
Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides,
Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla,
Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and
Fusobacterium.
[0065] "Gram positive bacteria" include cocci, nonsporulating rods,
and sporulating rods. The genera of gram positive bacteria include,
for example, Actinomyces, Bacillus, Clostridium, Corynebacterium,
Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,
Nocardia, Staphylococcus, Streptococcus, and Streptomyces.
[0066] The term "genus" is defined as a taxonomic group of related
species according to the Taxonomic Outline of Bacteria and Archaea
(Garrity, G. M., Lilbum, T. G., Cole, J. R., Harrison, S. H.,
Euzeby, J., and Tindall, B. J. (2007) The Taxonomic Outline of
Bacteria and Archaea. TOBA Release 7.7, March 2007. Michigan State
University Board of Trustees.
[0067] The term "species" is defined as a collection of closely
related organisms with greater than 97% 16S ribosomal RNA sequence
homology and greater than 70% genomic hybridization and
sufficiently different from all other organisms so as to be
recognized as a distinct unit.
[0068] The terms "recombinant microorganism," "modified
microorganism," and "recombinant host cell" are used
interchangeably herein and refer to microorganisms that have been
genetically modified to express or to overexpress endogenous
polynucleotides, to express heterologous polynucleotides, such as
those included in a vector, in an integration construct, or which
have an alteration in expression of an endogenous gene. By
"alteration" it is meant that the expression of the gene, or level
of a RNA molecule or equivalent RNA molecules encoding one or more
polypeptides or polypeptide subunits, or activity of one or more
polypeptides or polypeptide subunits is up regulated or down
regulated, such that expression, level, or activity is greater than
or less than that observed in the absence of the alteration. For
example, the term "alter" can mean "inhibit," but the use of the
word "alter" is not limited to this definition. It is understood
that the terms "recombinant microorganism" and "recombinant host
cell" refer not only to the particular recombinant microorganism
but to the progeny or potential progeny of such a microorganism.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term as used herein.
[0069] The term "expression" with respect to a gene sequence refers
to transcription of the gene and, as appropriate, translation of
the resulting mRNA transcript to a protein. Thus, as will be clear
from the context, expression of a protein results from
transcription and translation of the open reading frame sequence.
The level of expression of a desired product in a host cell may be
determined on the basis of either the amount of corresponding mRNA
that is present in the cell, or the amount of the desired product
encoded by the selected sequence. For example, mRNA transcribed
from a selected sequence can be quantitated by qRT-PCR or by
Northern hybridization (see Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
Protein encoded by a selected sequence can be quantitated by
various methods, e.g., by ELISA, by assaying for the biological
activity of the protein, or by employing assays that are
independent of such activity, such as western blotting or
radioimmunoassay, using antibodies that recognize and bind the
protein. See Sambrook et al., 1989, supra.
[0070] The term "overexpression" refers to an elevated level (e.g.,
aberrant level) of mRNAs encoding for a protein(s), and/or to
elevated levels of protein(s) in cells as compared to similar
corresponding unmodified cells expressing basal levels of mRNAs or
having basal levels of proteins. In particular embodiments, mRNA(s)
or protein(s) may be overexpressed by at least 2-fold, 3-fold,
4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold or more
in microorganisms engineered to exhibit increased gene mRNA,
protein, and/or activity.
[0071] As used herein and as would be understood by one of ordinary
skill in the art, "reduced activity and/or expression" of a protein
such as an enzyme can mean either a reduced specific catalytic
activity of the protein (e.g. reduced activity) and/or decreased
concentrations of the protein in the cell (e.g. reduced
expression). As would be understood by one or ordinary skill in the
art, the reduced activity of a protein in a cell may result from
decreased concentrations of the protein in the cell.
[0072] The term "wild-type microorganism" describes a cell that
occurs in nature, i.e. a cell that has not been genetically
modified. A wild-type microorganism can be genetically modified to
express or overexpress a first target enzyme. This microorganism
can act as a parental microorganism in the generation of a
microorganism modified to express or overexpress a second target
enzyme. In turn, the microorganism modified to express or
overexpress a first and a second target enzyme can be modified to
express or overexpress a third target enzyme.
[0073] Accordingly, a "parental microorganism" functions as a
reference cell for successive genetic modification events. Each
modification event can be accomplished by introducing a nucleic
acid molecule in to the reference cell. The introduction
facilitates the expression or overexpression of a target enzyme. It
is understood that the term "facilitates" encompasses the
activation of endogenous polynucleotides encoding a target enzyme
through genetic modification of e.g., a promoter sequence in a
parental microorganism. It is further understood that the term
"facilitates" encompasses the introduction of heterologous
polynucleotides encoding a target enzyme in to a parental
microorganism
[0074] The term "engineer" refers to any manipulation of a
microorganism that results in a detectable change in the
microorganism, wherein the manipulation includes but is not limited
to inserting a polynucleotide and/or polypeptide heterologous to
the microorganism and mutating a polynucleotide and/or polypeptide
native to the microorganism.
[0075] The term "mutation" as used herein indicates any
modification of a nucleic acid and/or polypeptide which results in
an altered nucleic acid or polypeptide. Mutations include, for
example, point mutations, deletions, or insertions of single or
multiple residues in a polynucleotide, which includes alterations
arising within a protein-encoding region of a gene as well as
alterations in regions outside of a protein-encoding sequence, such
as, but not limited to, regulatory or promoter sequences. A genetic
alteration may be a mutation of any type. For instance, the
mutation may constitute a point mutation, a frame-shift mutation, a
nonsense mutation, an insertion, or a deletion of part or all of a
gene. In addition, in some embodiments of the modified
microorganism, a portion of the microorganism genome has been
replaced with a heterologous polynucleotide. In some embodiments,
the mutations are naturally-occurring. In other embodiments, the
mutations are identified and/or enriched through artificial
selection pressure. In still other embodiments, the mutations in
the microorganism genome are the result of genetic engineering.
[0076] The term "biosynthetic pathway", also referred to as
"metabolic pathway", refers to a set of anabolic or catabolic
biochemical reactions for converting one chemical species into
another. Gene products belong to the same "metabolic pathway" if
they, in parallel or in series, act on the same substrate, produce
the same product, or act on or produce a metabolic intermediate
(i.e., metabolite) between the same substrate and metabolite end
product.
[0077] As used herein, the term "isobutanol producing metabolic
pathway" refers to an enzyme pathway which produces isobutanol from
pyruvate.
[0078] The term "NADH-dependent" as used herein with reference to
an enzyme, e.g., KARI and/or ADH, refers to an enzyme that
catalyzes the reduction of a substrate coupled to the oxidation of
NADH with a catalytic efficiency that is greater than the reduction
of the same substrate coupled to the oxidation of NADPH at equal
substrate and cofactor concentrations.
[0079] The term "exogenous" as used herein with reference to
various molecules, e.g., polynucleotides, polypeptides, enzymes,
etc., refers to molecules that are not normally or naturally found
in and/or produced by a given yeast, bacterium, organism,
microorganism, or cell in nature.
[0080] On the other hand, the term "endogenous" or "native" as used
herein with reference to various molecules, e.g., polynucleotides,
polypeptides, enzymes, etc., refers to molecules that are normally
or naturally found in and/or produced by a given yeast, bacterium,
organism, microorganism, or cell in nature.
[0081] The term "heterologous" as used herein in the context of a
modified host cell refers to various molecules, e.g.,
polynucleotides, polypeptides, enzymes, etc., wherein at least one
of the following is true: (a) the molecule(s) is/are foreign
("exogenous") to (i.e., not naturally found in) the host cell; (b)
the molecule(s) is/are naturally found in (e.g., is "endogenous
to") a given host microorganism or host cell but is either produced
in an unnatural location or in an unnatural amount in the cell;
and/or (c) the molecule(s) differ(s) in nucleotide or amino acid
sequence from the endogenous nucleotide or amino acid sequence(s)
such that the molecule differing in nucleotide or amino acid
sequence from the endogenous nucleotide or amino acid as found
endogenously is produced in an unnatural (e.g., greater than
naturally found) amount in the cell.
[0082] The term "feedstock" is defined as a raw material or mixture
of raw materials supplied to a microorganism or fermentation
process from which other products can be made. For example, a
carbon source, such as biomass or the carbon compounds derived from
biomass are a feedstock for a microorganism that produces a biofuel
in a fermentation process. However, a feedstock may contain
nutrients other than a carbon source.
[0083] The term "substrate" or "suitable substrate" refers to any
substance or compound that is converted or meant to be converted
into another compound by the action of an enzyme. The term includes
not only a single compound, but also combinations of compounds,
such as solutions, mixtures and other materials which contain at
least one substrate, or derivatives thereof. Further, the term
"substrate" encompasses not only compounds that provide a carbon
source suitable for use as a starting material, such as any biomass
derived sugar, but also intermediate and end product metabolites
used in a pathway associated with a recombinant microorganism as
described herein.
[0084] The term "fermentation" or "fermentation process" is defined
as a process in which a microorganism is cultivated in a culture
medium containing raw materials, such as feedstock and nutrients,
wherein the microorganism converts raw materials, such as a
feedstock, into products.
[0085] The term "volumetric productivity" or "production rate" is
defined as the amount of product formed per volume of medium per
unit of time. Volumetric productivity is reported in gram per liter
per hour (g/L/h).
[0086] The term "specific productivity" or "specific production
rate" is defined as the amount of product formed per volume of
medium per unit of time per amount of cells. Specific productivity
is reported in gram (or milligram) per gram cell dry weight per
hour (g/g h).
[0087] The term "yield" is defined as the amount of product
obtained per unit weight of raw material and may be expressed as g
product per g substrate (g/g). Yield may be expressed as a
percentage of the theoretical yield. "Theoretical yield" is defined
as the maximum amount of product that can be generated per a given
amount of substrate as dictated by the stoichiometry of the
metabolic pathway used to make the product. For example, the
theoretical yield for one typical conversion of glucose to
isobutanol is 0.41 g/g. As such, a yield of isobutanol from glucose
of 0.39 gig would be expressed as 95% of theoretical or 95%
theoretical yield.
[0088] The term "titer" is defined as the strength of a solution or
the concentration of a substance in solution. For example, the
titer of a biofuel in a fermentation broth is described as g of
biofuel in solution per liter of fermentation broth (g/L).
[0089] "Aerobic conditions" are defined as conditions under which
the oxygen concentration in the fermentation medium is sufficiently
high for an aerobic or facultative anaerobic microorganism to use
as a terminal electron acceptor.
[0090] In contrast, "anaerobic conditions" are defined as
conditions under which the oxygen concentration in the fermentation
medium is too low for the microorganism to use as a terminal
electron acceptor. Anaerobic conditions may be achieved by sparging
a fermentation medium with an inert gas such as nitrogen until
oxygen is no longer available to the microorganism as a terminal
electron acceptor. Alternatively, anaerobic conditions may be
achieved by the microorganism consuming the available oxygen of the
fermentation until oxygen is unavailable to the microorganism as a
terminal electron acceptor. Methods for the production of
isobutanol under anaerobic conditions are described in commonly
owned and co-pending publication, US 2010/0143997, the disclosures
of which are herein incorporated by reference in its entirety for
all purposes.
[0091] "Aerobic metabolism" refers to a biochemical process in
which oxygen is used as a terminal electron acceptor to make
energy, typically in the form of ATP, from carbohydrates. Aerobic
metabolism occurs, e.g., via glycolysis and the TCA cycle, wherein
a single glucose molecule is metabolized completely into carbon
dioxide in the presence of oxygen.
[0092] In contrast, "anaerobic metabolism" refers to a biochemical
process in which oxygen is not the final acceptor of electrons
contained in NADH. Anaerobic metabolism can be divided into
anaerobic respiration, in which compounds other than oxygen serve
as the terminal electron acceptor, and substrate level
phosphorylation, in which the electrons from NADH are utilized to
generate a reduced product via a "fermentative pathway."
[0093] In "fermentative pathways", NAD(P)H donates its electrons to
a molecule produced by the same metabolic pathway that produced the
electrons carried in NAD(P)H. For example, in one of the
fermentative pathways of certain yeast strains, NAD(P)H generated
through glycolysis transfers its electrons to pyruvate, yielding
ethanol. Fermentative pathways are usually active under anaerobic
conditions but may also occur under aerobic conditions, under
conditions where NADH is not fully oxidized via the respiratory
chain. For example, above certain glucose concentrations, Crabtree
positive yeasts produce large amounts of ethanol under aerobic
conditions.
[0094] The term "byproduct" or "by-product" means an undesired
product related to the production of an amino acid, amino acid
precursor, chemical, chemical precursor, biofuel, biofuel
precursor, higher alcohol, or higher alcohol precursor.
[0095] The term "substantially free" when used in reference to the
presence or absence of a protein activity (3-KAR enzymatic
activity, ALDH enzymatic activity, PDC enzymatic activity. GPD
enzymatic activity, etc.) means the level of the protein is
substantially less than that of the same protein in the wild-type
host, wherein less than about 50% of the wild-type level is
preferred and less than about 30% is more preferred. The activity
may be less than about 20%, less than about 10%, less than about
5%, or less than about 1% of wild-type activity. Microorganisms
which are "substantially free" of a particular protein activity
(3-KAR enzymatic activity, ALDH enzymatic activity, PDC enzymatic
activity, GPD enzymatic activity, etc.) may be created through
recombinant means or identified in nature.
[0096] The term "non-fermenting yeast" is a yeast species that
fails to demonstrate an anaerobic metabolism in which the electrons
from NADH are utilized to generate a reduced product via a
fermentative pathway such as the production of ethanol and CO.sub.2
from glucose. Non-fermentative yeast can be identified by the
"Durham Tube Test" (J. A. Bamett, R. W. Payne, and D. Yarrow. 2000.
Yeasts Characteristics and Identification. 3.sup.rd edition. p.
28-29. Cambridge University Press, Cambridge, UK) or by monitoring
the production of fermentation productions such as ethanol and
CO.sub.2.
[0097] The term "polynucleotide" is used herein interchangeably
with the term "nucleic acid" and refers to an organic polymer
composed of two or more monomers including nucleotides, nucleosides
or analogs thereof, including but not limited to single stranded or
double stranded, sense or antisense deoxyribonucleic acid (DNA) of
any length and, where appropriate, single stranded or double
stranded, sense or antisense ribonucleic acid (RNA) of any length,
including siRNA. The term "nucleotide" refers to any of several
compounds that consist of a ribose or deoxyribose sugar joined to a
purine or a pyrimidine base and to a phosphate group, and that are
the basic structural units of nucleic acids. The term "nucleoside"
refers to a compound (as guanosine or adenosine) that consists of a
purine or pyrimidine base combined with deoxyribose or ribose and
is found especially in nucleic acids. The term "nucleotide analog"
or "nucleoside analog" refers, respectively, to a nucleotide or
nucleoside in which one or more individual atoms have been replaced
with a different atom or with a different functional group.
Accordingly, the term polynucleotide includes nucleic acids of any
length, DNA, RNA, analogs and fragments thereof. A polynucleotide
of three or more nucleotides is also called nucleotidic oligomer or
oligonucleotide.
[0098] It is understood that the polynucleotides described herein
include "genes" and that the nucleic acid molecules described
herein include "vectors" or "plasmids." Accordingly, the term
"gene", also called a "structural gene" refers to a polynucleotide
that codes for a particular sequence of amino acids, which comprise
all or part of one or more proteins or enzymes, and may include
regulatory (non-transcribed) DNA sequences, such as promoter
sequences, which determine for example the conditions under which
the gene is expressed. The transcribed region of the gene may
include untranslated regions, including introns, 5'-untranslated
region (UTR), and 3'-UTR, as well as the coding sequence.
[0099] The term "operon" refers to two or more genes which are
transcribed as a single transcriptional unit from a common
promoter. In some embodiments, the genes comprising the operon are
contiguous genes. It is understood that transcription of an entire
operon can be modified (i.e., increased, decreased, or eliminated)
by modifying the common promoter. Alternatively, any gene or
combination of genes in an operon can be modified to alter the
function or activity of the encoded polypeptide. The modification
can result in an increase in the activity of the encoded
polypeptide. Further, the modification can impart new activities on
the encoded polypeptide. Exemplary new activities include the use
of alternative substrates and/or the ability to function in
alternative environmental conditions.
[0100] A "vector" is any means by which a nucleic acid can be
propagated and/or transferred between organisms, cells, or cellular
components. Vectors include viruses, bacteriophage, pro-viruses,
plasmids, phagemids, transposons, and artificial chromosomes such
as YACs (yeast artificial chromosomes), BACs (bacterial artificial
chromosomes), and PLACs (plant artificial chromosomes), and the
like, that are "episomes," that is, that replicate autonomously or
can integrate into a chromosome of a host cell. A vector can also
be a naked RNA polynucleotide, a naked DNA polynucleotide, a
polynucleotide composed of both DNA and RNA within the same strand,
a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or
RNA, a liposome-conjugated DNA, or the like, that are not episomal
in nature, or it can be an organism which comprises one or more of
the above polynucleotide constructs such as an agrobacterium or a
bacterium.
[0101] "Transformation" refers to the process by which a vector is
introduced into a host cell. Transformation (or transduction, or
transfection), can be achieved by any one of a number of means
including chemical transformation (e.g. lithium acetate
transformation), electroporation, microinjection, biolistics (or
particle bombardment-mediated delivery), or agrobacterium mediated
transformation.
[0102] The term "enzyme" as used herein refers to any substance
that catalyzes or promotes one or more chemical or biochemical
reactions, which usually includes enzymes totally or partially
composed of a polypeptide or polypeptides, but can include enzymes
composed of a different molecule including polynucleotides.
[0103] The term "protein," "peptide," or "polypeptide" as used
herein indicates an organic polymer composed of two or more amino
acidic monomers and/or analogs thereof. As used herein, the term
"amino acid" or "amino acidic monomer" refers to any natural and/or
synthetic amino acids including glycine and both D or L optical
isomers. The term "amino acid analog" refers to an amino acid in
which one or more individual atoms have been replaced, either with
a different atom, or with a different functional group.
Accordingly, the term polypeptide includes amino acidic polymer of
any length including full length proteins, and peptides as well as
analogs and fragments thereof. A polypeptide of three or more amino
acids is also called a protein oligomer or oligopeptide
[0104] The term "homolog," used with respect to an original
polynucleotide or polypeptide of a first family or species, refers
to distinct polynucleotides or polypeptides of a second family or
species which are determined by functional, structural or genomic
analyses to be a polynucleotide or polypeptide of the second family
or species which corresponds to the original polynucleotide or
polypeptide of the first family or species. Most often, homologs
will have functional, structural or genomic similarities.
Techniques are known by which homologs of a polynucleotide or
polypeptide can readily be cloned using genetic probes and PCR.
Identity of cloned sequences as homolog can be confirmed using
functional assays and/or by genomic mapping of the genes.
[0105] A polypeptide has "homology" or is "homologous" to a second
polypeptide if the amino acid sequence encoded by a gene has a
similar amino acid sequence to that of the second gene.
Alternatively, a polypeptide has homology to a second polypeptide
if the two polypeptides have "similar" amino acid sequences. (Thus,
the terms "homologous polypeptides" or "homologous proteins" are
defined to mean that the two polypeptides have similar amino acid
sequences).
[0106] The term "analog" or "analogous" refers to polynucleotide or
polypeptide sequences that are related to one another in function
only and are not from common descent or do not share a common
ancestral sequence. Analogs may differ in sequence but may share a
similar structure, due to convergent evolution. For example, two
enzymes are analogs or analogous if the enzymes catalyze the same
reaction of conversion of a substrate to a product, are unrelated
in sequence, and irrespective of whether the two enzymes are
related in structure.
Isobutanol Producing Recombinant Microorganisms
[0107] A variety of microorganisms convert sugars to produce
pyruvate, which is then utilized in a number of pathways of
cellular metabolism. In recent years, microorganisms, including
yeast, have been engineered to produce a number of desirable
products via pyruvate-driven biosynthetic pathways, including
isobutanol, an important commodity chemical and biofuel candidate
(See, e.g., commonly owned and co-pending patent publications, US
2009/0226991, US 2010/0143997, US 2011/0020889, US 2011/0076733,
and WO 2010/075504).
[0108] As described herein, the present invention relates to
recombinant microorganisms for producing isobutanol, wherein said
recombinant microorganisms comprise an isobutanol producing
metabolic pathway. In one embodiment, the isobutanol producing
metabolic pathway to convert pyruvate to isobutanol can be
comprised of the following reactions:
[0109] 1. 2 pyruvate.fwdarw.acetolactate+CO.sub.2
[0110] 2.
acetolactate+NAD(P)H.fwdarw.2,3-dihydroxyisovalerate+NAD(P).sup.-
+
[0111] 3. 2,3-dihydroxyisovalerate.fwdarw.alpha-ketoisovalerate
[0112] 4.
alpha-ketoisovalerate.fwdarw.isobutyraldehyde+CO.sub.2
[0113] 5. isobutyraldehyde+NAD(P)H.fwdarw.isobutanol+NADP
[0114] In one embodiment, these reactions are carried out by the
enzymes 1) Acetolactate synthase (ALS), 2) Ketol-acid
reductoisomerase (KARI), 3) Dihydroxy-acid dehydratase (DHAD), 4)
2-keto-acid decarboxylase, e.g., Keto-isovalerate decarboxylase
(KIVD), and 5) an Alcohol dehydrogenase (ADH) (FIG. 1). In some
embodiments, the recombinant microorganism may be engineered to
overexpress one or more of these enzymes. In an exemplary
embodiment, the recombinant microorganism is engineered to
overexpress all of these enzymes.
[0115] Alternative pathways for the production of isobutanol in
yeast have been described in WO/2007/050671 and in Dickinson et
al., 1998, J Biol Chem 273:25751-6. These and other isobutanol
producing metabolic pathways are within the scope of the present
application. In one embodiment, the isobutanol producing metabolic
pathway comprises five substrate to product reactions. In another
embodiment, the isobutanol producing metabolic pathway comprises
six substrate to product reactions. In yet another embodiment, the
isobutanol producing metabolic pathway comprises seven substrate to
product reactions.
[0116] In various embodiments described herein, the recombinant
microorganism comprises an isobutanol producing metabolic pathway.
In one embodiment, the isobutanol producing metabolic pathway
comprises at least one exogenous gene encoding a polypeptide that
catalyzes a step in the conversion of pyruvate to isobutanol. In
another embodiment, the isobutanol producing metabolic pathway
comprises at least two exogenous genes encoding polypeptides that
catalyze steps in the conversion of pyruvate to isobutanol. In yet
another embodiment, the isobutanol producing metabolic pathway
comprises at least three exogenous genes encoding polypeptides that
catalyze steps in the conversion of pyruvate to isobutanol. In yet
another embodiment, the isobutanol producing metabolic pathway
comprises at least four exogenous genes encoding polypeptides that
catalyze steps in the conversion of pyruvate to isobutanol. In yet
another embodiment, the isobutanol producing metabolic pathway
comprises at least five exogenous genes encoding polypeptides that
catalyze steps in the conversion of pyruvate to isobutanol. In yet
another embodiment, all of the isobutanol producing metabolic
pathway steps in the conversion of pyruvate to isobutanol are
converted by exogenously encoded enzymes.
[0117] In one embodiment, one or more of the isobutanol pathway
genes encodes an enzyme that is localized to the cytosol. In one
embodiment, the recombinant microorganisms comprise an isobutanol
producing metabolic pathway with at least one isobutanol pathway
enzyme localized in the cytosol. In another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least two isobutanol pathway enzymes
localized in the cytosol. In yet another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least three isobutanol pathway enzymes
localized in the cytosol. In yet another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least four isobutanol pathway enzymes
localized in the cytosol. In an exemplary embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with five isobutanol pathway enzymes localized in
the cytosol. In yet another exemplary embodiment, the recombinant
microorganisms comprise an isobutanol producing metabolic pathway
with all isobutanol pathway enzymes localized in the cytosol.
Isobutanol producing metabolic pathways in which one or more genes
are localized to the cytosol are described in commonly owned and
co-pending U.S. application Ser. No. 12/855,276, which is herein
incorporated by reference in its entirety for all purposes.
[0118] As is understood in the art, a variety of organisms can
serve as sources for the isobutanol pathway enzymes, including, but
not limited to, Saccharomyces spp., including S. cerevisiae and S.
uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis,
and K. marxianus, Pichia spp., Hansenula spp., including H.
polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp.,
including Y. spp. stipitis, Torulaspora pretoriensis, Issatchenkia
orientalis, Schizosaccharomyces spp., including S. pombe,
Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustilago
spp. Sources of genes from anaerobic fungi include, but not limited
to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp.
Sources of prokaryotic enzymes that are useful include, but not
limited to, Escherichia spp., Zymomonas spp., Staphylococcus spp.,
Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas
spp., Lactococcus spp., Enterobacter spp., Streptococcus spp.,
Salmonella spp., Slackia spp., Cryptobacterium spp., and
Eggerthella spp.
[0119] In some embodiments, one or more of these enzymes can be
encoded by native genes. Alternatively, one or more of these
enzymes can be encoded by heterologous genes.
[0120] For example, acetolactate synthases capable of converting
pyruvate to acetolactate may be derived from a variety of sources
(e.g., bacterial, yeast, Archaea, etc.), including B. subtilis
(GenBank Accession No. Q04789.3), L. lactis (GenBank Accession No.
NP.sub.--267340.1), S. mutans (GenBank Accession No.
NP.sub.--721805.1), K. pneumoniae (GenBank Accession No.
ZP.sub.--06014957.1), C. glutamicum (GenBank Accession No.
P42463.1), E. cloacae (GenBank Accession No. YP.sub.--003613611.1),
M. maripaludis (GenBank Accession No. ABX01060.1), M. grisea
(GenBank Accession No. AAB81248.1), T. stipitatus (GenBank
Accession No. XP.sub.--002485976.1), or S. cerevisiae ILV2 (GenBank
Accession No. NP.sub.--013826.1). Additional acetolactate synthases
capable of converting pyruvate to acetolactate are described in
commonly owned and co-pending US Publication No. 2011/0076733,
which is herein incorporated by reference in its entirety. A review
article characterizing the biosynthesis of acetolactate from
pyruvate via the activity of acetolactate synthases is provided by
Chipman et al., 1998, Biochimica et Biophysica Acta 1385: 401-19,
which is herein incorporated by reference in its entirety. Chipman
et al. provide an alignment and consensus for the sequences of a
representative number of acetolactate synthases. Motifs shared in
common between the majority of acetolactate synthases include:
TABLE-US-00001 (SEQ ID NO: 65) SGPG(A/C/V)(T/S)N, (SEQ ID NO: 66)
GX(P/A)GX(V/A/T), (SEQ ID NO: 67)
GX(Q/G)(T/A)(L/M)G(Y/F/W)(A/G)X(P/G) (W/A)AX(G/T)(A/V), and (SEQ ID
NO: 68) GD(G/A)(G/S/C)F
motifs at amino acid positions corresponding to the 163-169,
240-245, 521-535, and 549-553 residues, respectively, of the S.
cerevisiae ILV2. Thus, a protein harboring one or more of these
amino acid motifs can generally be expected to exhibit acetolactate
synthase activity.
[0121] Dihydroxy acid dehydratases capable of converting
2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate may be derived
from a variety of sources (e.g., bacterial, yeast, Archaea, etc.),
including E. coli (GenBank Accession No. YP.sub.--026248.1), L.
lactis (GenBank Accession No. NP.sub.--267379.1), S. mutans
(GenBank Accession No. NP.sub.--722414.1), M. stadtmanae (GenBank
Accession No. YP.sub.--448586.1), M. tractuosa (GenBank Accession
No. YP.sub.--004053736.1), Eubacterium SCB49 (GenBank Accession No.
ZP.sub.--01890126.1), G. forsetti (GenBank Accession No.
YP.sub.--862145.1), Y. lipolytica (GenBank Accession No.
XP.sub.--502180.2), N. crassa (GenBank Accession No.
XP.sub.--963045.1), or S. cerevisiae ILV3 (GenBank Accession No.
NP.sub.--012550.1). Additional dihydroxy acid dehydratases capable
of 2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate are
described in commonly owned and co-pending US Publication No.
2011/0076733. Motifs shared in common between the majority of
dihydroxy acid dehydratases include:
TABLE-US-00002 (SEQ ID NO: 69) SLXSRXXIA, (SEQ ID NO: 70) CDKXXPG,
(SEQ ID NO: 71) GXCXGXXTAN, (SEQ ID NO: 72) GGSTN, (SEQ ID NO: 73)
GPXGXPGMRXE, (SEQ ID NO: 74) ALXTDGRXSG, and (SEQ ID NO: 75)
GHXXPEA
motifs at amino acid positions corresponding to the 93-101,
122-128, 193-202, 276-280, 482-491, 509-518, and 526-532 residues,
respectively, of the E. coli dihydroxy acid dehydratase encoded by
ilvD. Thus, a protein harboring one or more of these amino acid
motifs can generally be expected to exhibit dihydroxy acid
dehydratase activity.
[0122] 2-keto-acid decarboxylases capable of converting
.alpha.-ketoisovalerate to isobutyraldehyde may be derived from a
variety of sources (e.g., bacterial, yeast, Archaea, etc.),
including L. lactis kivD (GenBank Accession No.
YP.sub.--003353820.1), E. cloacae (GenBank Accession No. P23234.1),
M. smegmatis (GenBank Accession No. A0R480.1), M. tuberculosis
(GenBank Accession No. 053865.1), M. avium (GenBank Accession No.
Q742Q2.1, A. brasilense (GenBank Accession No. P51852.1), L. lactis
kdcA (GenBank Accession No. AAS49166.1), S. epidermidis (GenBank
Accession No. NP.sub.--765765.1), M. caseolyticus (GenBank
Accession No. YP.sub.--002560734.1), B. megaterium (GenBank
Accession No. YP.sub.--003561644.1), S. cerevisiae ARO10 (GenBank
Accession No. NP.sub.--010668.1), or S. cerevisiae THI3 (GenBank
Accession No. CAA98646.1). Additional 2-keto-acid decarboxylases
capable of converting .alpha.-ketoisovalerate to isobutyraldehyde
are described in commonly owned and co-pending US Publication No.
2011/0076733. Motifs shared in common between the majority of
2-keto-acid decarboxylases include:
TABLE-US-00003 (SEQ ID NO: 76) FG(V/I)(P/S)G(D/E)(Y/F), (SEQ ID NO:
77) (T/V)T(F/Y)G(V/A)G(E/A)(L/F)(S/N), (SEQ ID NO: 78)
N(G/A)(L/I/V)AG(S/A)(Y/F)AE, (SEQ ID NO: 79)
(V/I)(L/I/V)XI(V/T/S)G, and (SEQ ID NO: 80)
GDG(S/A)(L/F/A)Q(L/M)T
motifs at amino acid positions corresponding to the 21-27, 70-78,
81-89, 93-98, and 428-435 residues, respectively, of the L. lactis
2-keto-acid decarboxylase encoded by kivD. Thus, a protein
harboring one or more of these amino acid motifs can generally be
expected to exhibit 2-keto-acid decarboxylase activity.
[0123] Alcohol dehydrogenases capable of converting
isobutyraldehyde to isobutanol may be derived from a variety of
sources (e.g., bacterial, yeast, Archaea, etc.), including L.
lactis (GenBank Accession No. YP.sub.--003354381), B. cereus
(GenBank Accession No. YP.sub.--001374103.1), N. meningitidis
(GenBank Accession No. CBA03965.1), S. sanguinis (GenBank Accession
No. YP.sub.--001035842.1), L. brevis (GenBank Accession No.
YP.sub.--794451.1), B. thuringiensis (GenBank Accession No.
ZP.sub.--04101989.1), P. acidilactici (GenBank Accession No.
ZP.sub.--06197454.1), B. subtilis (GenBank Accession No.
EHA31115.1), N. crassa (GenBank Accession No. CAB91241.1) or S.
cerevisiae ADH6 (GenBank Accession No. NP.sub.--014051.1).
Additional alcohol dehydrogenases capable of converting
isobutyraldehyde to isobutanol are described in commonly owned and
co-pending US Publication Nos. 2011/0076733 and 2011/0201072.
Motifs shared in common between the majority of alcohol
dehydrogenases include:
TABLE-US-00004 (SEQ ID NO: 81) C(H/G)(T/S)D(L/I)H, (SEQ ID NO: 82)
GHEXXGXV, (SEQ ID NO: 83) (L/V)(Q/K/E)(V/I/K)G(D/Q)(R/H)(V/A), (SEQ
ID NO: 84) CXXCXXC, (SEQ ID NO: 85) (C/A)(A/G/D)(G/A)XT(T/V), and
(SEQ ID NO: 86) G(L/A/C)G(G/P)(L/I/V)G
motifs at amino acid positions corresponding to the 39-44, 59-66,
76-82, 91-97, 147-152, and 171-176 residues, respectively, of the
L. lactis alcohol dehydrogenase encoded by adhA. Thus, a protein
harboring one or more of these amino acid motifs can generally be
expected to exhibit alcohol dehydrogenase activity.
[0124] In another embodiment, the yeast microorganism may be
engineered to have increased ability to convert pyruvate to
isobutanol. In one embodiment, the yeast microorganism may be
engineered to have increased ability to convert pyruvate to
isobutyraldehyde. In another embodiment, the yeast microorganism
may be engineered to have increased ability to convert pyruvate to
keto-isovalerate. In another embodiment, the yeast microorganism
may be engineered to have increased ability to convert pyruvate to
2,3-dihydroxyisovalerate. In another embodiment, the yeast
microorganism may be engineered to have increased ability to
convert pyruvate to acetolactate.
[0125] Furthermore, any of the genes encoding the foregoing enzymes
(or any others mentioned herein (or any of the regulatory elements
that control or modulate expression thereof)) may be optimized by
genetic/protein engineering techniques, such as directed evolution
or rational mutagenesis, which are known to those of ordinary skill
in the art. Such action allows those of ordinary skill in the art
to optimize the enzymes for expression and activity in yeast.
Isobutanol-Producing Metabolic Pathways with Improved KARI
Properties
[0126] As described herein, the present application provides
several KARI enzymes that give high performance when expressed in
yeast in the context of isobutanol production. Accordingly, this
application describes methods of increasing isobutanol production
through the use of recombinant microorganisms comprising KARI
enzymes with improved properties for the production of
isobutanol.
[0127] One aspect of the application is directed to an isolated
nucleic acid molecule encoding a ketol-acid reductoisomerase
(KARI), wherein said KARI is at least about 80% identical to SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ
ID NO; 28, SEQ ID NO: 40, or SEQ ID NO: 56. Further within the
scope of present application are KARIs which are at least about
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6. SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO; 28, SEQ ID NO: 40, or SEQ ID NO:
56.
[0128] In one embodiment, the KARI is derived from the genus
Shewanella. In a specific embodiment, the KARI is derived from
Shewanella sp. strain MR-4. In another specific embodiment, the
isolated nucleic acid molecule is comprised of SEQ ID NO: 1. In
another embodiment, the KARI is derived from the genus Vibrio. In a
specific embodiment, the KARI is derived from Vibrio fischeri. In
another specific embodiment, the isolated nucleic acid molecule is
comprised of SEQ ID NO: 3. In yet another embodiment, the KARI is
derived from the genus Gramella. In a specific embodiment, the KARI
is derived from Gramella forsetii. In another specific embodiment,
the isolated nucleic acid molecule is comprised of SEQ ID NO: 5. In
yet another embodiment, the KARI is derived from the genus
Cytophaga. In a specific embodiment, the KARI is derived from
Cytophaga hutchinsonii. In another specific embodiment, the
isolated nucleic acid molecule is comprised of SEQ ID NO: 7. In yet
another embodiment, the KARI is derived from a genus selected from
Lactococcus and Streptococcus. In a specific embodiment, the KARI
is derived from Lactococcus lactis, Streptococcus equinus, or
Streptococcus infantarius. In another specific embodiment, the KARI
is encoded by SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
or SEQ ID NO: 25. In yet another embodiment, the KARI is derived
from the genus Methanococcus. In a specific embodiment, the KARI is
derived from Methanococcus maripaludis, Methanococcus vannielii, or
Methanococcus voltae. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, or SEQ ID NO: 37. In yet another embodiment, the
KARI is derived from a genus selected from Zymomonas,
Erythrobacter, Sphingomonas, Sphingobium, and Novosphingobium. In a
specific embodiment, the KARI is derived from Zymomonas mobilis,
Erythrobacter litoralis, Sphingomonas wittichii, Sphingobium
japonicum, Sphingobium chlorophenolicum, or Novosphingobium
nitrogenifigens. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:
45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO: 53.
In yet another embodiment, the KARI is derived from the genus
Bacteroides. In a specific embodiment, the KARI is derived from
Bacteroides thetaiotaomicron. In another specific embodiment, the
KARI is encoded by SEQ ID NO: 55. In yet another embodiment, the
KARI is derived from the genus Schizosaccharomyces. In a specific
embodiment, the KARI is derived from Schizosaccharomyces pombe or
Schizosaccharomyces japonicus. In another specific embodiment, the
KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO:
61.
[0129] Also included within the scope of this application are
isolated KARI enzymes that have been modified to be NADH-dependent.
Accordingly, the present application further relates to
NADH-dependent ketol-acid reductoisomerases (NKRs) derived from a
KARI that is at least about 80% identical to SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28,
SEQ ID NO: 40, or SEQ ID NO: 58.
[0130] Another aspect of the application is directed to an isolated
nucleic acid molecule encoding a ketol-acid reductoisomerase
(KARI), wherein said KARI is at least about 99% identical to SEQ ID
NO: 64. In one embodiment, the KARI is derived from the genus
Salmonella. In a specific embodiment, the KARI is derived from
Salmonella enterica. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 63. The present application further relates
to NADH-dependent ketol-acid reductoisomerases (NKRs) derived from
a KARI that is at least about 99% identical to SEQ ID NO: 64.
[0131] The invention also includes fragments of the disclosed KARI
enzymes which comprise at least 50, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, or 600 amino acid residues and retain one or
more activities associated with KARI enzymes. Such fragments may be
obtained by deletion mutation, by recombinant techniques that are
routine and well-known in the art, or by enzymatic digestion of the
KARI enzyme(s) of interest using any of a number of well-known
proteolytic enzymes. The invention further includes nucleic acid
molecules which encode the above described KARI enzymes and KARI
enzyme fragments.
[0132] Another aspect of the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID
NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO; 28, SEQ ID NO: 40,
or SEQ ID NO: 56. Further within the scope of present application
are recombinant microorganism comprising at least one nucleic acid
molecule encoding a ketol-acid reductoisomerase (KARI), wherein
said KARI is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO; 28, SEQ ID NO:
40, or SEQ ID NO: 56.
[0133] In one embodiment, the KARI is derived from the genus
Shewanella. In a specific embodiment, the KARI is derived from
Shewanella sp. strain MR-4. In another specific embodiment, the
isolated nucleic acid molecule is comprised of SEQ ID NO: 1. In
another embodiment, the KARI is derived from the genus Vibrio. In a
specific embodiment, the KARI is derived from Vibrio fischeri. In
another specific embodiment, the isolated nucleic acid molecule is
comprised of SEQ ID NO: 3. In yet another embodiment, the KARI is
derived from the genus Gramella. In a specific embodiment, the KARI
is derived from Gramella forsetii. In another specific embodiment,
the isolated nucleic acid molecule is comprised of SEQ ID NO: 5. In
yet another embodiment, the KARI is derived from the genus
Cytophaga. In a specific embodiment, the KARI is derived from
Cytophaga hutchinsonii. In another specific embodiment, the
isolated nucleic acid molecule is comprised of SEQ ID NO: 7. In yet
another embodiment, the KARI is derived from a genus selected from
Lactococcus and Streptococcus. In a specific embodiment, the KARI
is derived from Lactococcus lactis, Streptococcus equinus, or
Streptococcus infantarius. In another specific embodiment, the KARI
is encoded by SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
or SEQ ID NO: 25. In yet another embodiment, the KARI is derived
from the genus Methanococcus. In a specific embodiment, the KARI is
derived from Methanococcus maripaludis, Methanococcus vannielii, or
Methanococcus voltae. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, or SEQ ID NO: 37. In yet another embodiment, the
KARI is derived from a genus selected from Zymomonas,
Erythrobacter, Sphingomonas, Sphingobium, and Novosphingobium. In a
specific embodiment, the KARI is derived from Zymomonas mobilis,
Erythrobacter litoralis, Sphingomonas wittichii, Sphingobium
japonicum, Sphingobium chlorophenolicum, or Novosphingobium
nitrogenifigens. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:
45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO: 53.
In yet another embodiment, the KARI is derived from the genus
Bacteroides. In a specific embodiment, the KARI is derived from
Bacteroides thetaiotaomicron. In another specific embodiment, the
KARI is encoded by SEQ ID NO: 55. In yet another embodiment, the
KARI is derived from the genus Schizosaccharomyces. In a specific
embodiment, the KARI is derived from Schizosaccharomyces pombe or
Schizosaccharomyces japonicus. In another specific embodiment, the
KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO:
61.
[0134] Another aspect of the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 99% identical to SEQ ID NO: 64. In one embodiment,
the KARI is derived from the genus Salmonella. In a specific
embodiment, the KARI is derived from Salmonella enterica. In
another specific embodiment, the KARI is encoded by SEQ ID NO:
63.
[0135] In an exemplary embodiment, pathway steps 2 and 5 of the
isobutanol pathway may be carried out by KARI and ADH enzymes that
utilize NADH (rather than NADPH) as a cofactor. It has been found
previously that utilization of NADH-dependent KARI (NKR) and ADH
enzymes to catalyze pathway steps 2 and 5, respectively,
surprisingly enables production of isobutanol at theoretical yield
and/or under anaerobic conditions. See, e.g., commonly owned and
co-pending patent publication US 2010/0143997. An example of an
NADH-dependent isobutanol pathway is illustrated in FIG. 2. Thus,
in one embodiment, the recombinant microorganisms of the present
invention may use an NKR to catalyze the conversion of acetolactate
to produce 2,3-dihydroxyisovalerate. In another embodiment, the
recombinant microorganisms of the present invention may use an
NADH-dependent ADH to catalyze the conversion of isobutyraldehyde
to produce isobutanol. In yet another embodiment, the recombinant
microorganisms of the present invention may use both an NKR to
catalyze the conversion of acetolactate to produce
2,3-dihydroxyisovalerate, and an NADH-dependent ADH to catalyze the
conversion of isobutyraldehyde to produce isobutanol.
[0136] In an exemplary embodiment, the NKR is derived from a KARI
that is at least about 80% identical to SEQ ID NO: 2. In another
exemplary embodiment, the NKR is a KARI enzyme that has one or more
modifications or mutations at positions corresponding to amino
acids selected from: (a) alanine 71 of the Shewanella sp. KARI (SEQ
ID NO: 2); (b) arginine 76 of the Shewanella sp. KARI (SEQ ID NO:
2); (c) serine 78 of the Shewanella sp. KARI; and (d) glutamine 110
of the Shewanella sp. KARI (SEQ ID NO: 2).
[0137] In one specific embodiment, the application is directed to
KARI enzymes wherein the alanine corresponding to position 71 of
the Shewanella sp. KARI (SEQ ID NO: 2) is replaced with an amino
acid selected from serine, threonine, asparagine, or glutamine. In
another specific embodiment, the application is directed to KARI
enzymes wherein the arginine corresponding to position 76 of the
Shewanella sp. KARI (SEQ ID NO: 2) is replaced with aspartic acid
or glutamic acid. In yet another specific embodiment, the
application is directed to KARI enzymes wherein the serine
corresponding to position 78 of the Shewanella sp. KARI (SEQ ID NO:
2) is replaced with aspartic acid or glutamic acid. In yet another
specific embodiment, the application is directed to KARI enzymes
wherein the glutamine corresponding to position 110 of the
Shewanella sp. KARI (SEQ ID NO: 2) is replaced with valine,
alanine, isoleucine, leucine, methionine, phenylalanine,
tryptophan, or tyrosine.
[0138] In another specific embodiment, the application relates to a
KARI enzyme having four modifications or mutations at positions
corresponding to amino acids selected from: (a) alanine 71 of the
Shewanella sp. KARI (SEQ ID NO: 2); (b) arginine 76 of the
Shewanella sp. KARI (SEQ ID NO: 2); (c) serine 78 of the Shewanella
sp. KARI; and (d) glutamine 110 of the Shewanella sp. KARI (SEQ ID
NO: 2).
[0139] In another exemplary embodiment, the NKR is derived from a
KARI that is at least about 80% identical to SEQ ID NO: 10. In
another exemplary embodiment, the NKR is a KARI enzyme that has one
or more modifications or mutations at positions corresponding to
amino acids selected from: (a) valine 48 of the L. lactis KARI (SEQ
ID NO: 10); (b) arginine 49 of the L. lactis KARI (SEQ ID NO: 10);
(c) lysine 52 of the L. lactis KARI (SEQ ID NO: 10); (d) serine 53
of the L. lactis KARI (SEQ ID NO: 10); (e) glutamic acid 59 of the
L. lactis KARI (SEQ ID NO: 10): (f); leucine 85 of the L. lactis
KARI (SEQ ID NO: 10); (g) isoleucine 89 of the L. lactis KARI (SEQ
ID NO: 10); (h) lysine 118 of the L. lactis KARI (SEQ ID NO: 10);
(i) threonine 182 of the L. lactis KARI (SEQ ID NO: 10); and (j)
glutamic acid 320 of the L. lactis KARI (SEQ ID NO: 10).
[0140] In one specific embodiment, the application is directed to
KARI enzymes wherein the valine corresponding to position 48 of the
L. lactis KARI (SEQ ID NO: 2) is replaced with leucine or proline.
In another specific embodiment, the application is directed to KARI
enzymes wherein the arginine corresponding to position 49 of the L.
lactis KARI (SEQ ID NO: 2) is replaced with valine, leucine,
serine, or proline. In yet another specific embodiment, the
application is directed to KARI enzymes wherein the lysine
corresponding to position 52 of the L. lactis KARI (SEQ ID NO: 2)
is replaced with leucine, alanine, isoleucine, methionine,
phenylalanine, tryptophan, tyrosine, valine, aspartic acid, or
glutamic acid. In yet another specific embodiment, the application
is directed to KARI enzymes wherein the serine corresponding to
position 53 of the L. lactis KARI (SEQ ID NO: 2) is replaced with
aspartic acid or glutamic acid. In yet another specific embodiment,
the application is directed to KARI enzymes wherein the glutamic
acid corresponding to position 59 of the L. lactis KARI (SEQ ID NO:
2) is replaced with lysine, arginine, or histidine. In yet another
specific embodiment, the application is directed to KARI enzymes
wherein the leucine corresponding to position 85 of the L. lactis
KARI (SEQ ID NO: 2) is replaced with threonine or alanine. In yet
another specific embodiment, the application is directed to KARI
enzymes wherein the isoleucine corresponding to position 89 of the
L. lactis KARI (SEQ ID NO: 2) is replaced with alanine. In yet
another specific embodiment, the application is directed to KARI
enzymes wherein the lysine corresponding to position 118 of the L.
lactis KARI (SEQ ID NO: 2) is replaced with glutamic acid or
aspartic acid. In yet another specific embodiment, the application
is directed to KARI enzymes wherein the threonine corresponding to
position 182 of the L. lactis KARI (SEQ ID NO: 2) is replaced with
serine, asparagine, or glutamine. In yet another specific
embodiment, the application is directed to KARI enzymes wherein the
glutamic acid corresponding to position 320 of the L. lactis KARI
(SEQ ID NO: 2) is replaced with lysine, arginine, or histidine.
[0141] In another specific embodiment, the application relates to a
KARI enzyme having seven modifications or mutations at positions
corresponding to amino acids selected from: (a) valine 48 of the L.
lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI
(SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO:
10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10): (e)
glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f)
threonine 182 of the L. lactis KARI (SEQ ID NO: 10); and (g)
glutamic acid 320 of the L. lactis KARI (SEQ ID NO: 10).
[0142] In yet another exemplary embodiment, the NKR is derived from
a KARI that is at least about 99% identical to SEQ ID NO: 64. In
another exemplary embodiment, the NKR is a KARI enzyme that has one
or more modifications or mutations at positions corresponding to
amino acids selected from: (a) alanine 71 of the S. enterica KARI
(SEQ ID NO: 64); (b) arginine 76 of the S. enterica KARI (SEQ ID
NO: 64); (c) serine 78 of the S. enterica KARI (SEQ ID NO: 64); (d)
glutamine 110 of the S. enterica KARI (SEQ ID NO: 64); (e) aspartic
acid 146 of the S. enterica KARI (SEQ ID NO: 64): (f) glycine 185
of the S. enterica KARI (SEQ ID NO: 64); and (g) lysine 433 of the
S. enterica KARI (SEQ ID NO: 64).
[0143] In one specific embodiment, the application is directed to
KARI enzymes wherein the alanine corresponding to position 71 of
the S. enterica KARI (SEQ ID NO: 64) is replaced with an amino acid
selected from serine, threonine, asparagine, or glutamine. In
another specific embodiment, the application is directed to KARI
enzymes wherein the arginine corresponding to position 76 of the S.
enterica KARI (SEQ ID NO: 64) is replaced with aspartic acid or
glutamic acid. In yet another specific embodiment, the application
is directed to KARI enzymes wherein the serine corresponding to
position 78 of the S. enterica KARI (SEQ ID NO: 64) is replaced
with aspartic acid or glutamic acid. In yet another specific
embodiment, the application is directed to KARI enzymes wherein the
glutamine corresponding to position 110 of the S. enterica KARI
(SEQ ID NO: 64) is replaced with valine, alanine, isoleucine,
leucine, methionine, phenylalanine, tryptophan, or tyrosine. In yet
another specific embodiment, the application is directed to KARI
enzymes wherein the aspartic acid corresponding to position 146 of
the S. enterica KARI (SEQ ID NO: 64) is replaced with glycine,
cysteine, or proline. In yet another specific embodiment, the
application is directed to KARI enzymes wherein the glycine
corresponding to position 185 of the S. enterica KARI (SEQ ID NO:
64) is replaced with arginine, histidine, or lysine. In yet another
specific embodiment, the application is directed to KARI enzymes
wherein the lysine corresponding to position 433 of the S. enterica
KARI (SEQ ID NO: 64) is replaced with glutamic acid or aspartic
acid.
[0144] In another specific embodiment, the application relates to a
KARI enzyme having seven modifications or mutations at positions
corresponding to amino acids selected from: (a) alanine 71 of the
S. enterica KARI (SEQ ID NO: 64); (b) arginine 76 of the S.
enterica KARI (SEQ ID NO: 64); (c) serine 78 of the S. enterica
KARI (SEQ ID NO: 64); (d) glutamine 110 of the S. enterica KARI
(SEQ ID NO: 64); (e) aspartic acid 146 of the S. enterica KARI (SEQ
ID NO: 64); (f) glycine 185 of the S. enterica KARI (SEQ ID NO:
64); and (g) lysine 433 of the S. enterica KARI (SEQ ID NO:
64).
[0145] Further included within the scope of the application are
KARI enzymes, other than the Shewanella sp. KARI (SEQ ID NO: 2),
the L. lactis KARI (SEQ ID NO: 10), or the S. enterica KARI (SEQ ID
NO: 64) which contain modifications or mutations corresponding to
those set out above. The nucleotide sequences for several KARI
enzymes are known. A representative listing of KARI enzymes capable
of being modified are disclosed in commonly owned and co-pending US
Publication No. US 2010/0143997.
[0146] The corresponding positions of the KARI enzyme identified
herein (e.g., the Shewanella sp. KARI, the L. lactis KARI, or the
S. enterica KARI) may be readily identified for other KARI enzymes
by one of skill in the art. Thus, given the defined region and the
assays described in the present application, one with skill in the
art can make one or a number of modifications which would result in
an increased ability to utilize NADH, particularly for the
conversion of acetolactate to 2,3-dihydroxyisovalerate, in any KARI
enzyme of interest. Residues to be modified in accordance with the
present application may include those described in Examples
4-5.
[0147] The application also includes fragments of the modified KARI
enzymes which comprise at least 50, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, or 600 amino acid residues and retain one or
more activities associated with KARI enzymes. Such fragments may be
obtained by deletion mutation, by recombinant techniques that are
routine and well-known in the art, or by enzymatic digestion of the
KARI enzyme(s) of interest using any of a number of well-known
proteolytic enzymes. The invention further includes nucleic acid
molecules which encode the above described mutant KARI enzymes and
KARI enzyme fragments.
[0148] Another aspect of the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI
has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) alanine 71 of the
Shewanella sp. KARI (SEQ ID NO: 2); (b) arginine 76 of the
Shewanella sp. KARI (SEQ ID NO: 2); (c) serine 78 of the Shewanella
sp. KARI; and (d) glutamine 110 of the Shewanella sp. KARI (SEQ ID
NO: 2). Further included within the scope of the application are
recombinant microorganisms comprising a KARI enzyme, other than the
Shewanella sp. KARI (SEQ ID NO: 2), which contains modifications or
mutations at positions corresponding to those set out above.
[0149] Yet another aspect of the application relates to a
recombinant microorganism comprising at least one nucleic acid
molecule encoding a ketol-acid reductoisomerase (KARI), wherein
said KARI has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) valine 48 of the L.
lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI
(SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO:
10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e)
glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f);
leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89
of the L. lactis KARI (SEQ ID NO: 10); (h) lysine 118 of the L.
lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L. lactis
KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis
KARI (SEQ ID NO: 10). Further included within the scope of the
application are recombinant microorganisms comprising a KARI
enzyme, other than the L. lactis KARI (SEQ ID NO: 10), which
contains modifications or mutations at positions corresponding to
those set out above.
[0150] Another aspect of the application relates to a recombinant
microorganism comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI
has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) alanine 71 of the
S. enterica KARI (SEQ ID NO: 64); (b) arginine 76 of the S.
enterica KARI (SEQ ID NO: 64); (c) serine 78 of the S. enterica
KARI (SEQ ID NO: 64); (d) glutamine 110 of the S. enterica KARI
(SEQ ID NO: 64); (e) aspartic acid 146 of the S. enterica KARI (SEQ
ID NO: 64); (f) glycine 185 of the S. enterica KARI (SEQ ID NO:
64); and (g) lysine 433 of the S. enterica KARI (SEQ ID NO: 64).
Further included within the scope of the application are
recombinant microorganisms comprising a KARI enzyme, other than the
S. enterica KARI (SEQ ID NO: 64), which contains modifications or
mutations at positions corresponding to those set out above.
[0151] Further within the scope of present application are
recombinant microorganisms comprising at least one nucleic acid
molecule encoding a ketol-acid reductoisomerase (KARI), wherein
said KARI is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO; 28, SEQ ID NO:
40, or SEQ ID NO: 56. Also within the scope of present application
are recombinant microorganisms comprising at least one nucleic acid
molecule encoding a ketol-acid reductoisomerase (KARI), wherein
said KARI is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to a KARI having one or more
modifications or mutations at positions corresponding to amino
acids selected from: (a) alanine 71 of the Shewanella sp. KARI (SEQ
ID NO: 2); (b) arginine 76 of the Shewanella sp. KARI (SEQ ID NO:
2); (c) serine 78 of the Shewanella sp. KARI; and (d) glutamine 110
of the Shewanella sp. KARI (SEQ ID NO: 2). Also within the scope of
present application are recombinant microorganisms comprising at
least one nucleic acid molecule encoding a ketol-acid
reductoisomerase (KARI), wherein said KARI is at least about 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to a KARI having one or more modifications or mutations at
positions corresponding to amino acids selected from: (a) valine 48
of the L. lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L.
lactis KARI (SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI
(SEQ ID NO: 10); (d) serine 53 of the L. lactis KARI (SEQ ID NO:
10); (e) glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10):
(f); leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g)
isoleucine 89 of the L. lactis KARI (SEQ ID NO: 10); (h) lysine 118
of the L. lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L.
lactis KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L.
lactis KARI (SEQ ID NO: 10).
[0152] Further within the scope of present application are
recombinant microorganisms comprising at least one nucleic acid
molecule encoding a ketol-acid reductoisomerase (KARI), wherein
said KARI is at least about 99% identical to SEQ ID NO: 64. Also
within the scope of present application are recombinant
microorganisms comprising at least one nucleic acid molecule
encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is
at least about 99% identical to a KARI having one or more
modifications or mutations at positions corresponding to amino
acids selected from: (a) alanine 71 of the S. enterica KARI (SEQ ID
NO: 64); (b) arginine 76 of the S. enterica KARI (SEQ ID NO: 64);
(c) serine 78 of the S. enterica KARI (SEQ ID NO: 64); (d)
glutamine 110 of the S. enterica KARI (SEQ ID NO: 64); (e) aspartic
acid 146 of the S. enterica KARI (SEQ ID NO: 64); (f) glycine 185
of the S. enterica KARI (SEQ ID NO: 64); and (g) lysine 433 of the
S. enterica KARI (SEQ ID NO: 64).
[0153] In accordance with the invention, any number of mutations
can be made to the KARI enzymes, and in a preferred aspect,
multiple mutations can be made to result in an increased ability to
utilize NADH for the conversion of acetolactate to
2,3-dihydroxyisovalerate. Such mutations include point mutations,
frame shift mutations, deletions, and insertions, with one or more
(e.g., one, two, three, four, five or more, etc.) point mutations
preferred.
[0154] Mutations may be introduced into the KARI enzymes of the
present application to create NKRs using any methodology known to
those skilled in the art. Mutations may be introduced randomly by,
for example, conducting a PCR reaction in the presence of manganese
as a divalent metal ion cofactor. Alternatively, oligonucleotide
directed mutagenesis may be used to create the NKRs which allows
for all possible classes of base pair changes at any determined
site along the encoding DNA molecule. In general, this technique
involves annealing an oligonucleotide complementary (except for one
or more mismatches) to a single stranded nucleotide sequence coding
for the KARI enzyme of interest. The mismatched oligonucleotide is
then extended by DNA polymerase, generating a double-stranded DNA
molecule which contains the desired change in sequence in one
strand. The changes in sequence can, for example, result in the
deletion, substitution, or insertion of an amino acid. The
double-stranded polynucleotide can then be inserted into an
appropriate expression vector, and a mutant or modified polypeptide
can thus be produced. The above-described oligonucleotide directed
mutagenesis can, for example, be carried out via PCR.
[0155] In one aspect, the NADH-dependent activity of the modified
or mutated KARI enzyme is increased.
[0156] In an exemplary embodiment, the catalytic efficiency of the
modified or mutated KARI enzyme is improved for the cofactor NADH.
Preferably, the catalytic efficiency of the modified or mutated
KARI enzyme is improved by at least about 5% as compared to the
wild-type or parental KARI for NADH. More preferably the catalytic
efficiency of the modified or mutated KARI enzyme is improved by at
least about 15% as compared to the wild-type or parental KARI for
NADH. More preferably, the catalytic efficiency of the modified or
mutated KARI enzyme is improved by at least about 25% as compared
to the wild-type or parental KARI for NADH. More preferably, the
catalytic efficiency of the modified or mutated KARI enzyme is
improved by at least about 50% as compared to the wild-type or
parental KARI for NADH. More preferably, the catalytic efficiency
of the modified or mutated KARI enzyme is improved by at least
about 75% as compared to the wild-type or parental KARI for NADH.
More preferably, the catalytic efficiency of the modified or
mutated KARI enzyme is improved by at least about 100% as compared
to the wild-type or parental KARI for NADH. More preferably, the
catalytic efficiency of the modified or mutated KARI enzyme is
improved by at least about 300% as compared to the wild-type or
parental KARI for NADH. More preferably, the catalytic efficiency
of the modified or mutated KARI enzyme is improved by at least
about 500% as compared to the wild-type or parental KARI for NADH.
More preferably, the catalytic efficiency of the modified or
mutated KARI enzyme is improved by at least about 1000% as compared
to the wild-type or parental KARI for NADH. More preferably, the
catalytic efficiency of the modified or mutated KARI enzyme is
improved by at least about 5000% as compared to the wild-type or
parental KARI for NADH.
[0157] In another exemplary embodiment, the catalytic efficiency of
the modified or mutated KARI enzyme with NADH is increased with
respect to the catalytic efficiency of the wild-type or parental
enzyme with NADPH. Preferably, the catalytic efficiency of the
modified or mutated KARI enzyme is at least about 10% of the
catalytic efficiency of the wild-type or parental KARI enzyme for
NADPH. More preferably, the catalytic efficiency of the modified or
mutated KARI enzyme is at least about 25% of the catalytic
efficiency of the wild-type or parental KARI enzyme for NADPH. More
preferably, the catalytic efficiency of the modified or mutated
KARI enzyme is at least about 50% of the catalytic efficiency of
the wild-type or parental KARI enzyme for NADPH. More preferably,
the catalytic efficiency of the modified or mutated KARI enzyme is
at least about 75%, 85%, 95% of the catalytic efficiency of the
wild-type or parental KARI enzyme for NADPH.
[0158] In another exemplary embodiment, the K.sub.M of the KARI
enzyme for NADH is decreased relative to the wild-type or parental
enzyme. A change in K.sub.M is evidenced by at least a 5% or
greater increase or decrease in K.sub.M compared to the wild-type
KARI enzyme. In certain embodiments, modified or mutated KARI
enzymes of the present invention may show greater than 10 times
decreased K.sub.M for NADH compared to the wild-type or parental
KARI enzyme. In certain embodiments, modified or mutated KARI
enzymes of the present invention may show greater than 30 times
decreased K.sub.M for NADH compared to the wild-type or parental
KARI enzyme.
[0159] In another exemplary embodiment, the k.sub.cat of the KARI
enzyme with NADH is increased relative to the wild-type or parental
enzyme. A change in k.sub.cat is evidenced by at least a 5% or
greater increase or decrease in K.sub.M compared to the wild-type
KARI enzyme. In certain embodiments, modified or mutated KARI
enzymes of the present invention may show greater than 50%
increased k.sub.cat for NADH compared to the wild-type or parental
KARI enzyme. In certain embodiments, modified or mutated KARI
enzymes of the present invention may show greater than 100%
increased k.sub.cat for NADH compared to the wild-type or parental
KARI enzyme. In certain embodiments, modified or mutated KARI
enzymes of the present invention may show greater than 200%
increased k.sub.cat for NADH compared to the wild-type or parental
KARI enzyme.
Recombinant Microorganisms Comprising KARI with Improved
Properties
[0160] In addition to isobutanol producing metabolic pathways, a
number of biosynthetic pathways use KARI enzymes to catalyze a
reaction step, including pathways for the production of isoleucine,
leucine, valine, pantothenate, coenzyme A, 1-butanol,
2-methyl-1-butanol, 3-methyl-1-butanol, 3-methyl-1-pentanol,
4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol. A
representative list of the engineered biosynthetic pathways
utilizing KARI enzymes is provided in Table 1.
TABLE-US-00005 TABLE 1 Biosynthetic Pathways Utilizing KARI to
Catalyze a Reaction Step. Biosynthetic Pathway Reference.sup.a
Isobutanol US 2009/0226991 (Feldman et al.), US 2011/0020889
(Feldman et al.), and US 2010/0143997 (Buelter et al.) Leucine
WO/2001/021772 (Yocum et al.) and McCourt et al., 2006, Amino Acids
31: 173-210 Valine WO/2001/021772 (Yocum et al.) and McCourt et
al., 2006, Amino Acids 31: 173-210 Pantothenic Acid WO/2001/021772
(Yocum et al.) Coenzyme A WO/2001/021772 (Yocum et al.) 1-Butanol
WO/2010/017230 (Lynch), WO/2010/031772 (Wu at al.), and
KR2011002130 (Lee et al.) 2-Methyl-1- WO/2008/098227 (Liao et al.),
WO/2009/076480 Butanol (Picataggio et al.), and Atsumi et al.,
2008, Nature 451: 86-89 3-Methyl-1- WO/2008/098227 (Liao et al.),
Atsumi et al., 2008, Butanol Nature 451: 86-89, and Connor et al.,
2008, Appl. Environ. Microbiol. 74: 5769-5775 3-Methyl-1-
WO/2010/045629 (Liao et al.), Zhang at al., 2008, Pentanol Proc
Natl Acad Sci USA 105: 20653-20658 4-Methyl-1- WO/2010/045629 (Liao
et al.), Zhang et al., 2008, Pentanol Proc Natl Acad Sci USA 105:
20653-20658 4-Methyl-1- WO/2010/045629 (Liao et al.), Zhang et al.,
2008, Hexanol Proc Natl Acad Sci USA 105:20653-20658 5-Methyl-1-
WO/2010/045629 (Liao et al.), Zhang et al., 2008, Heptanol Proc
Natl Acad Sci USA 105: 20653-20658 .sup.aThe contents of each of
the references in this table are herein incorporated by reference
in their entireties for all purposes.
[0161] As described above, each of these biosynthetic pathways uses
a KARI enzyme to catalyze a reaction step. Therefore, the product
yield from these biosynthetic pathways will in part depend upon the
activity of KARI.
[0162] As will be understood by one skilled in the art equipped
with the present disclosure, the KARI enzymes described herein
would have utility in any of the above-described pathways. Thus, in
an additional aspect, the present application relates to a
recombinant microorganism comprising a KARI-requiring biosynthetic
pathway, wherein said recombinant microorganism comprises at least
one nucleic acid molecule encoding a KARI that is at least about
80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID
NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO:
58. In one embodiment, the KARI is derived from the genus
Shewanella. In a specific embodiment, the KARI is derived from
Shewanella sp. strain MR-4. In another specific embodiment, the
isolated nucleic acid molecule is comprised of SEQ ID NO: 1. In
another embodiment, the KARI is derived from the genus Vibrio. In a
specific embodiment, the KARI is derived from Vibrio fischei. In
another specific embodiment, the isolated nucleic acid molecule is
comprised of SEQ ID NO: 3. In yet another embodiment, the KARI is
derived from the genus Gramella. In a specific embodiment, the KARI
is derived from Gramella forsetii. In another specific embodiment,
the isolated nucleic acid molecule is comprised of SEQ ID NO: 5. In
yet another embodiment, the KARI is derived from the genus
Cytophaga. In a specific embodiment, the KARI is derived from
Cytophaga hutchinsonii. In another specific embodiment, the
isolated nucleic acid molecule is comprised of SEQ ID NO: 7. In yet
another embodiment, the KARI is derived from a genus selected from
Lactococcus and Streptococcus. In a specific embodiment, the KARI
is derived from Lactococcus lactis, Streptococcus equinus, or
Streptococcus infantarius. In another specific embodiment, the KARI
is encoded by SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
or SEQ ID NO: 25. In yet another embodiment, the KARI is derived
from the genus Methanococcus. In a specific embodiment, the KARI is
derived from Methanococcus maripaludis, Methanococcus vannielii, or
Methanococcus voltae. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, or SEQ ID NO: 37. In yet another embodiment, the
KARI is derived from a genus selected from Zymomonas,
Erythrobacter, Sphingomonas, Sphingobium, and Novosphingobium. In a
specific embodiment, the KARI is derived from Zymomonas mobilis,
Erythrobacter litoralis, Sphingomonas wittichii, Sphingobium
japonicum, Sphingobium chlorophenolicum, or Novosphingobium
nitrogenifigens. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:
45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO: 53.
In yet another embodiment, the KARI is derived from the genus
Bacteroides. In a specific embodiment, the KARI is derived from
Bacteroides thetaiotaomicron. In another specific embodiment, the
KARI is encoded by SEQ ID NO: 55. In yet another embodiment, the
KARI is derived from the genus Schizosaccharomyces. In a specific
embodiment, the KARI is derived from Schizosaccharomyces pombe or
Schizosaccharomyces japonicus. In another specific embodiment, the
KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61.
In yet another embodiment, the KARI has one or more modifications
or mutations at positions corresponding to amino acids selected
from: (a) alanine 71 of the Shewanella sp. KARI (SEQ ID NO: 2); (b)
arginine 76 of the Shewanella sp. KARI (SEQ ID NO: 2); (c) serine
78 of the Shewanella sp. KARI; and (d) glutamine 110 of the
Shewanella sp. KARI (SEQ ID NO: 2). In yet another embodiment, the
KARI has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) valine 48 of the L.
lactis KARI (SEQ ID NO: 10): (b) arginine 49 of the L. lactis KARI
(SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO:
10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e)
glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f);
leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89
of the L. lactis KARI (SEQ ID NO: 10); (h) lysine 118 of the L.
lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L. lactis
KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis
KARI (SEQ ID NO: 10).
[0163] In an additional aspect, the present application relates to
a recombinant microorganism comprising a KARI-requiring
biosynthetic pathway, wherein said recombinant microorganism
comprises at least one nucleic acid molecule encoding a KARI that
is at least about 99% identical to SEQ ID NO: 64. In one
embodiment, the KARI is derived from the genus Salmonella. In a
specific embodiment, the KARI is derived from Salmonella enterica.
In another specific embodiment, the KARI is encoded by SEQ ID NO:
63. In yet another embodiment, the KARI has one or more
modifications or mutations at positions corresponding to amino
acids selected from: (a) alanine 71 of the S. enterica KARI (SEQ ID
NO: 64); (b) arginine 76 of the S. enterica KARI (SEQ ID NO: 64);
(c) serine 78 of the S. enterica KARI (SEQ ID NO: 64); (d)
glutamine 110 of the S. enterica KARI (SEQ ID NO: 64); (e) aspartic
acid 146 of the S. enterica KARI (SEQ ID NO: 64); (f) glycine 185
of the S. enterica KARI (SEQ ID NO: 64); and (g) lysine 433 of the
S. enterica KARI (SEQ ID NO: 64).
[0164] As used herein, a "KARI-requiring biosynthetic pathway"
refers to any metabolic pathway which utilizes KARI to convert
acetolactate to 2,3-dihydroxyisovalerate or
2-aceto-2-hydroxy-butanoate to 2,3-dihydroxy-3-methylvalerate.
Examples of KARI-requiring biosynthetic pathways include, but are
not limited to, isobutanol, isoleucine, leucine, valine,
pantothenate, coenzyme A, 1-butanol, 2-methyl-1-butanol,
3-methyl-1-butanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol,
4-methyl-1-hexanol, and 5-methyl-1-heptanol metabolic pathways. The
metabolic pathway may naturally occur in a microorganism (e.g., a
natural pathway for the production of valine) or arise from the
introduction of one or more heterologous polynucleotides through
genetic engineering. In an exemplary embodiment, the recombinant
microorganisms expressing the KARI-requiring biosynthetic pathway
are yeast cells.
The Microorganism in General
[0165] As described herein, the recombinant microorganisms of the
present invention can express a plurality of heterologous and/or
native enzymes involved in pathways for the production of a
beneficial metabolite such as isobutanol.
[0166] As described herein, "engineered" or "modified"
microorganisms are produced via the introduction of genetic
material into a host or parental microorganism of choice and/or by
modification of the expression of native genes, thereby modifying
or altering the cellular physiology and biochemistry of the
microorganism. Through the introduction of genetic material and/or
the modification of the expression of native genes the parental
microorganism acquires new properties, e.g., the ability to produce
a new, or greater quantities of, an intracellular and/or
extracellular metabolite. As described herein, the introduction of
genetic material into and/or the modification of the expression of
native genes in a parental microorganism results in a new or
modified ability to produce beneficial metabolites such as
isobutanol from a suitable carbon source. The genetic material
introduced into and/or the genes modified for expression in the
parental microorganism contains gene(s), or parts of genes, coding
for one or more of the enzymes involved in a biosynthetic pathway
for the production of isobutanol and may also include additional
elements for the expression and/or regulation of expression of
these genes, e.g., promoter sequences.
[0167] In addition to the introduction of a genetic material into a
host or parental microorganism, an engineered or modified
microorganism can also include the alteration, disruption, deletion
or knocking-out of a gene or polynucleotide to alter the cellular
physiology and biochemistry of the microorganism. Through the
alteration, disruption, deletion or knocking-out of a gene or
polynucleotide, the microorganism acquires new or improved
properties (e.g., the ability to produce a new metabolite or
greater quantities of an intracellular metabolite, to improve the
flux of a metabolite down a desired pathway, and/or to reduce the
production of by-products).
[0168] Recombinant microorganisms provided herein may also produce
metabolites in quantities not available in the parental
microorganism. A "metabolite" refers to any substance produced by
metabolism or a substance necessary for or taking part in a
particular metabolic process. A metabolite can be an organic
compound that is a starting material (e.g., glucose or pyruvate),
an intermediate (e.g., 2-ketoisovalerate), or an end product (e.g.,
isobutanol) of metabolism. Metabolites can be used to construct
more complex molecules, or they can be broken down into simpler
ones. Intermediate metabolites may be synthesized from other
metabolites, perhaps used to make more complex substances, or
broken down into simpler compounds, often with the release of
chemical energy.
[0169] The disclosure identifies specific genes useful in the
methods, compositions and organisms of the disclosure; however it
will be recognized that absolute identity to such genes is not
necessary. For example, changes in a particular gene or
polynucleotide comprising a sequence encoding a polypeptide or
enzyme can be performed and screened for activity. Typically such
changes comprise conservative mutations and silent mutations. Such
modified or mutated polynucleotides and polypeptides can be
screened for expression of a functional enzyme using methods known
in the art.
[0170] Due to the inherent degeneracy of the genetic code, other
polynucleotides which encode substantially the same or functionally
equivalent polypeptides can also be used to clone and express the
polynucleotides encoding such enzymes.
[0171] As will be understood by those of skill in the art, it can
be advantageous to modify a coding sequence to enhance its
expression in a particular host. The genetic code is redundant with
64 possible codons, but most organisms typically use a subset of
these codons. The codons that are utilized most often in a species
are called optimal codons, and those not utilized very often are
classified as rare or low-usage codons. Codons can be substituted
to reflect the preferred codon usage of the host, in a process
sometimes called "codon optimization" or "controlling for species
codon bias."
[0172] Optimized coding sequences containing codons preferred by a
particular prokaryotic or eukaryotic host (Murray et al., 1989,
Nucl Acids Res. 17: 477-508) can be prepared, for example, to
increase the rate of translation or to produce recombinant RNA
transcripts having desirable properties, such as a longer
half-life, as compared with transcripts produced from a
non-optimized sequence. Translation stop codons can also be
modified to reflect host preference. For example, typical stop
codons for S. cerevisiae and mammals are UAA and UGA, respectively.
The typical stop codon for monocotyledonous plants is UGA, whereas
insects and E. coli commonly use UAA as the stop codon (Dalphin et
al., 1996, Nucl Acids Res. 24: 216-8). Methodology for optimizing a
nucleotide sequence for expression in a plant is provided, for
example, in U.S. Pat. No. 6,015,891, and the references cited
therein.
[0173] Those of skill in the art will recognize that, due to the
degenerate nature of the genetic code, a variety of DNA compounds
differing in their nucleotide sequences can be used to encode a
given enzyme of the disclosure. The native DNA sequence encoding
the biosynthetic enzymes described above are referenced herein
merely to illustrate an embodiment of the disclosure, and the
disclosure includes DNA compounds of any sequence that encode the
amino acid sequences of the polypeptides and proteins of the
enzymes utilized in the methods of the disclosure. In similar
fashion, a polypeptide can typically tolerate one or more amino
acid substitutions, deletions, and insertions in its amino acid
sequence without loss or significant loss of a desired activity.
The disclosure includes such polypeptides with different amino acid
sequences than the specific proteins described herein so long as
the modified or variant polypeptides have the enzymatic anabolic or
catabolic activity of the reference polypeptide. Furthermore, the
amino acid sequences encoded by the DNA sequences shown herein
merely illustrate embodiments of the disclosure.
[0174] In addition, homologs of enzymes useful for generating
metabolites are encompassed by the microorganisms and methods
provided herein.
[0175] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences have at
least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine
the percent identity of two amino acid sequences, or of two nucleic
acid sequences, the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second amino acid or nucleic acid sequence for optimal
alignment and non-homologous sequences can be disregarded for
comparison purposes). In one embodiment, the length of a reference
sequence aligned for comparison purposes is at least 30%, typically
at least 40%, more typically at least 50%, even more typically at
least 60%, and even more typically at least 70%, 80%, 90%, 100% of
the length of the reference sequence. The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position (as used herein amino acid or
nucleic acid "identity" is equivalent to amino acid or nucleic acid
"homology"). The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences.
[0176] When "homologous" is used in reference to proteins or
peptides, it is recognized that residue positions that are not
identical often differ by conservative amino acid substitutions. A
"conservative amino acid substitution" is one in which an amino
acid residue is substituted by another amino acid residue having a
side chain (R group) with similar chemical properties (e.g., charge
or hydrophobicity). In general, a conservative amino acid
substitution will not substantially change the functional
properties of a protein. In cases where two or more amino acid
sequences differ from each other by conservative substitutions, the
percent sequence identity or degree of homology may be adjusted
upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well known to those of skill
in the art (See, e.g., Pearson W. R., 1994, Methods in Mol Biol 25:
365-89).
[0177] The following six groups each contain amino acids that are
conservative substitutions for one another: 1) Serine (S),
Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3)
Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Alanine (A), Valine (V), and 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0178] Sequence homology for polypeptides, which is also referred
to as percent sequence identity, is typically measured using
sequence analysis software. See commonly owned and co-pending
application US 2009/0226991. A typical algorithm used comparing a
molecule sequence to a database containing a large number of
sequences from different organisms is the computer program BLAST.
When searching a database containing sequences from a large number
of different organisms, it is typical to compare amino acid
sequences. Database searching using amino acid sequences can be
measured by algorithms described in commonly owned U.S. Pat. No.
8,017,375.
[0179] It is understood that a range of microorganisms can be
modified to include an isobutanol producing metabolic pathway
suitable for the production of isobutanol. In various embodiments,
the microorganisms may be selected from yeast microorganisms. Yeast
microorganisms for the production of isobutanol may be selected
based on certain characteristics:
[0180] One characteristic may include the property that the
microorganism is selected to convert various carbon sources into
isobutanol. The term "carbon source" generally refers to a
substance suitable to be used as a source of carbon for prokaryotic
or eukaryotic cell growth. Examples of suitable carbon sources are
described in commonly owned U.S. Pat. No. 8,017,375. Accordingly,
in one embodiment, the recombinant microorganism herein disclosed
can convert a variety of carbon sources to products, including but
not limited to glucose, galactose, mannose, xylose, arabinose,
lactose, sucrose, CO.sub.2, and mixtures thereof.
[0181] The recombinant microorganism may thus further include a
pathway for the production of isobutanol from five-carbon (pentose)
sugars including xylose. Most yeast species metabolize xylose via a
complex route, in which xylose is first reduced to xylitol via a
xylose reductase (XR) enzyme. The xylitol is then oxidized to
xylulose via a xylitol dehydrogenase (XDH) enzyme. The xylulose is
then phosphorylated via a xylulokinase (XK) enzyme. This pathway
operates inefficiently in yeast species because it introduces a
redox imbalance in the cell. The xylose-to-xylitol step uses
primarily NADPH as a cofactor (generating NADP+), whereas the
xylitol-to-xylulose step uses NAD+ as a cofactor (generating NADH).
Other processes must operate to restore the redox imbalance within
the cell. This often means that the organism cannot grow
anaerobically on xylose or other pentose sugars. Accordingly, a
yeast species that can efficiently ferment xylose and other pentose
sugars into a desired fermentation product is therefore very
desirable.
[0182] Thus, in one aspect, the recombinant microorganism is
engineered to express a functional exogenous xylose isomerase.
Exogenous xylose isomerases (XI) functional in yeast are known in
the art. See, e.g., Rajgarhia et al., U.S. Pat. No. 7,943,366,
which is herein incorporated by reference in its entirety. In an
embodiment according to this aspect, the exogenous XI gene is
operatively linked to promoter and terminator sequences that are
functional in the yeast cell. In a preferred embodiment, the
recombinant microorganism further has a deletion or disruption of a
native gene that encodes for an enzyme (e.g., XR and/or XDH) that
catalyzes the conversion of xylose to xylitol. In a further
preferred embodiment, the recombinant microorganism also contains a
functional, exogenous xylulokinase (XK) gene operatively linked to
promoter and terminator sequences that are functional in the yeast
cell. In one embodiment, the xylulokinase (XK) gene is
overexpressed.
[0183] In one embodiment, the yeast microorganism has reduced or no
pyruvate decarboxylase (PDC) activity. PDC catalyzes the
decarboxylation of pyruvate to acetaldehyde, which is then reduced
to ethanol by ADH via an oxidation of NADH to NAD+. Ethanol
production is the main pathway to oxidize the NADH from glycolysis.
Deletion, disruption, or mutation of this pathway increases the
pyruvate and the reducing equivalents (NADH) available for a
biosynthetic pathway which uses pyruvate as the starting material
and/or as an intermediate. Accordingly, deletion, disruption, or
mutation of one or more genes encoding for pyruvate decarboxylase
and/or a positive transcriptional regulator thereof can further
increase the yield of the desired pyruvate-derived metabolite
(e.g., isobutanol). In one embodiment, said pyruvate decarboxylase
gene targeted for disruption, deletion, or mutation is selected
from the group consisting of PDC1, PDC5, and PDC6, or homologs or
variants thereof. In another embodiment, all three of PDC1, PDC5,
and PDC6 are targeted for disruption, deletion, or mutation. In yet
another embodiment, a positive transcriptional regulator of the
PDC1, PDC5, and/or PDC6 is targeted for disruption, deletion or
mutation. In one embodiment, said positive transcriptional
regulator is PDC2, or homologs or variants thereof.
[0184] As is understood by those skilled in the art, there are
several additional mechanisms available for reducing or disrupting
the activity of a protein encoded by PDC1, PDC5, PDC6, and/or PDC2,
including, but not limited to, the use of a regulated promoter, use
of a weak constitutive promoter, disruption of one of the two
copies of the gene in a diploid yeast, disruption of both copies of
the gene in a diploid yeast, expression of an anti-sense nucleic
acid, expression of an siRNA, over expression of a negative
regulator of the endogenous promoter, alteration of the activity of
an endogenous or heterologous gene, use of a heterologous gene with
lower specific activity, the like or combinations thereof. Yeast
strains with reduced PDC activity are described in commonly owned
U.S. Pat. No. 8,017,375, as well as commonly owned and co-pending
US Patent Publication No. 2011/0183392.
[0185] In another embodiment, the microorganism has reduced
glycerol-3-phosphate dehydrogenase (GPD) activity. GPD catalyzes
the reduction of dihydroxyacetone phosphate (DHAP) to
glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+.
Glycerol is then produced from G3P by Glycerol-3-phosphatase (GPP).
Glycerol production is a secondary pathway to oxidize excess NADH
from glycolysis. Reduction or elimination of this pathway would
increase the pyruvate and reducing equivalents (NADH) available for
the production of a pyruvate-derived metabolite (e.g., isobutanol).
Thus, disruption, deletion, or mutation of the genes encoding for
glycerol-3-phosphate dehydrogenases can further increase the yield
of the desired metabolite (e.g., isobutanol). Yeast strains with
reduced GPD activity are described in commonly owned and co-pending
US Patent Publication Nos. 2011/0020889 and 2011/0183392.
[0186] In yet another embodiment, the microorganism has reduced
3-keto acid reductase (3-KAR) activity. 3-KARs catalyze the
conversion of 3-keto acids (e.g., acetolactate) to 3-hydroxyacids
(e.g., DH2 MB). Yeast strains with reduced 3-KAR activity are
described in commonly owned U.S. Pat. Nos. 8,133,715, 8,153,415,
and 8,158,404, which are herein incorporated by reference in their
entireties.
[0187] In yet another embodiment, the microorganism has reduced
aldehyde dehydrogenase (ALDH) activity. Aldehyde dehydrogenases
catalyze the conversion of aldehydes (e.g., isobutyraldehyde) to
acid by-products (e.g., isobutyrate). Yeast strains with reduced
ALDH activity are described in commonly owned U.S. Pat. Nos.
8,133,715, 8,153,415, and 8,158,404, which are herein incorporated
by reference in their entireties.
[0188] In one embodiment, the yeast microorganisms may be selected
from the "Saccharomyces Yeast Clade", as described in commonly
owned U.S. Pat. No. 8,017,375.
[0189] The term "Saccharomyces sensu stricto" taxonomy group is a
cluster of yeast species that are highly related to S. cerevisiae
(Rainieri et al., 2003, J. Biosci Bioengin 96: 1-9). Saccharomyces
sensu stricto yeast species include but are not limited to S.
cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S.
carocanis and hybrids derived from these species (Masneuf et al.,
1998, Yeast 7: 61-72).
[0190] An ancient whole genome duplication (WGD) event occurred
during the evolution of the hemiascomycete yeast and was discovered
using comparative genomic tools (Kellis et al., 2004, Nature 428:
617-24; Dujon et al., 2004, Nature 430:35-44; Langkjaer et al.,
2003, Nature 428: 848-52; Wolfe et al., 1997, Nature 387: 708-13).
Using this major evolutionary event, yeast can be divided into
species that diverged from a common ancestor following the WGD
event (termed "post-WGD yeast" herein) and species that diverged
from the yeast lineage prior to the WGD event (termed "pre-WGD
yeast" herein).
[0191] Accordingly, in one embodiment, the yeast microorganism may
be selected from a post-WGD yeast genus, including but not limited
to Saccharomyces and Candida. The favored post-WGD yeast species
include: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S.
castelli, and C. glabrata.
[0192] In another embodiment, the yeast microorganism may be
selected from a pre-whole genome duplication (pre-WGD) yeast genus
including but not limited to Saccharomyces, Kluyveromyces, Candida,
Pichia, Issatchenkia, Debaryomyces, Hansenula, Yarrowia and,
Schizosaccharomyces. Representative pre-WGD yeast species include:
S. kluyveri, K. thermotolerans, K. marxianus, K. waltii, K. lactis,
C. tropicalis, P. pastoris, P. anomala, P. stipitis, I. orientalis,
I. occidentalis, I. scutulata, D. hansenii, H. anomala, Y.
lipolytica, and S. pombe.
[0193] A yeast microorganism may be either Crabtree-negative or
Crabtree-positive as described in described in commonly owned U.S.
Pat. No. 8,017,375. In one embodiment the yeast microorganism may
be selected from yeast with a Crabtree-negative phenotype including
but not limited to the following genera: Saccharomyces,
Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida.
Crabtree-negative species include but are not limited to: S.
kluyvenri, K. lactis, K. marxianus, P. anomala, P. stipitis, I.
orientalis, I. occidentalis, I. scutulata, H. anomala, and C.
utilis. In another embodiment, the yeast microorganism may be
selected from yeast with a Crabtree-positive phenotype, including
but not limited to Saccharomyces, Kluyveromyces, Zygosaccharomyces,
Debaryomyces, Pichia and Schizosaccharomyces. Crabtree-positive
yeast species include but are not limited to: S. cerevisiae, S.
uvarum, S. bayanus, S. paradoxus, S. castelli, K. thermotolerans,
C. glabrata, Z. bailli, Z. rouxii, D. hansenii, P. pastorius, and
S. pombe.
[0194] Another characteristic may include the property that the
microorganism is that it is non-fermenting. In other words, it
cannot metabolize a carbon source anaerobically while the yeast is
able to metabolize a carbon source in the presence of oxygen.
Nonfermenting yeast refers to both naturally occurring yeasts as
well as genetically modified yeast. During anaerobic fermentation
with fermentative yeast, the main pathway to oxidize the NADH from
glycolysis is through the production of ethanol. Ethanol is
produced by alcohol dehydrogenase (ADH) via the reduction of
acetaldehyde, which is generated from pyruvate by pyruvate
decarboxylase (PDC). In one embodiment, a fermentative yeast can be
engineered to be non-fermentative by the reduction or elimination
of the native PDC activity. Thus, most of the pyruvate produced by
glycolysis is not consumed by PDC and is available for the
isobutanol pathway. Deletion of this pathway increases the pyruvate
and the reducing equivalents available for the biosynthetic
pathway. Fermentative pathways contribute to low yield and low
productivity of pyruvate-derived metabolites such as isobutanol.
Accordingly, deletion of one or more PDC genes may increase yield
and productivity of a desired metabolite (e.g., isobutanol).
[0195] In some embodiments, the recombinant microorganisms may be
microorganisms that are non-fermenting yeast microorganisms,
including, but not limited to those, classified into a genera
selected from the group consisting of Tricosporon, Rhodotorula,
Myxozyma, or Candida. In a specific embodiment, the non-fermenting
yeast is C. xestobii.
Methods in General
Identification of KARI Homologs
[0196] Any method can be used to identify genes that encode for
enzymes that are homologous to the genes described herein (e.g.,
KARI homologs). Generally, genes that are homologous or similar to
the KARIs described herein may be identified by functional,
structural, and/or genetic analysis. In most cases, homologous or
similar genes and/or homologous or similar enzymes will have
functional, structural, or genetic similarities.
[0197] Techniques known to those skilled in the art may be suitable
to identify additional homologous genes and homologous enzymes.
Generally, analogous genes and/or analogous enzymes can be
identified by functional analysis and will have functional
similarities. Techniques known to those skilled in the art may be
suitable to identify analogous genes and analogous enzymes. For
example, to identify homologous or analogous genes, proteins, or
enzymes, techniques may include, but not limited to, cloning a gene
by PCR using primers based on a published sequence of a gene/enzyme
or by degenerate PCR using degenerate primers designed to amplify a
conserved region among ketol-acid reductoisomerase genes. Further,
one skilled in the art can use techniques to identify homologous or
analogous genes, proteins, or enzymes with functional homology or
similarity. Techniques include examining a cell or cell culture for
the catalytic activity of an enzyme through in vitro enzyme assays
for said activity (e.g. as described herein or in Kiritani, K.
Branched-Chain Amino Acids Methods Enzymology, 1970), then
isolating the enzyme with said activity through purification,
determining the protein sequence of the enzyme through techniques
such as Edman degradation, design of PCR primers to the likely
nucleic acid sequence, amplification of said DNA sequence through
PCR, and cloning of said nucleic acid sequence. To identify
homologous or similar genes and/or homologous or similar enzymes,
analogous genes and/or analogous enzymes or proteins, techniques
also include comparison of data concerning a candidate gene or
enzyme with databases such as BRENDA, KEGG, or MetaCYC. The
candidate gene or enzyme may be identified within the above
mentioned databases in accordance with the teachings herein.
Genetic Insertions and Deletions
[0198] Any method can be used to introduce a nucleic acid molecule
into yeast and many such methods are well known. For example,
transformation and electroporation are common methods for
introducing nucleic acid into yeast cells. See, e.g., Gietz et al.,
1992, Nuc Acids Res. 27: 69-74; Ito et al., 1983, J. Bacteriol.
153: 163-8; and Becker et al., 1991, Methods in Enzymology 194:
182-7.
[0199] In an embodiment, the integration of a gene of interest into
a DNA fragment or target gene of a yeast microorganism occurs
according to the principle of homologous recombination. According
to this embodiment, an integration cassette containing a module
comprising at least one yeast marker gene and/or the gene to be
integrated (internal module) is flanked on either side by DNA
fragments homologous to those of the ends of the targeted
integration site (recombinogenic sequences). After transforming the
yeast with the cassette by appropriate methods, a homologous
recombination between the recombinogenic sequences may result in
the internal module replacing the chromosomal region in between the
two sites of the genome corresponding to the recombinogenic
sequences of the integration cassette. (Orr-Weaver et al., 1981,
PNAS USA 78: 6354-58).
[0200] In an embodiment, the integration cassette for integration
of a gene of interest into a yeast microorganism includes the
heterologous gene under the control of an appropriate promoter and
terminator together with the selectable marker flanked by
recombinogenic sequences for integration of a heterologous gene
into the yeast chromosome. In an embodiment, the heterologous gene
includes an appropriate native gene desired to increase the copy
number of a native gene(s). The selectable marker gene can be any
marker gene used in yeast, including but not limited to, HIS3,
TRP1, LEU2, URA3, bar, ble, hph, and kan. The recombinogenic
sequences can be chosen at will, depending on the desired
integration site suitable for the desired application.
[0201] In another embodiment, integration of a gene into the
chromosome of the yeast microorganism may occur via random
integration (Kooistra et al., 2004, Yeast 21: 781-792).
[0202] Additionally, in an embodiment, certain introduced marker
genes are removed from the genome using techniques well known to
those skilled in the art. For example, URA3 marker loss can be
obtained by plating URA3 containing cells in FOA (5-fluoro-orotic
acid) containing medium and selecting for FOA resistant colonies
(Boeke et al., 1984, Mol. Gen. Genet 197: 345-47).
[0203] The exogenous nucleic acid molecule contained within a yeast
cell of the disclosure can be maintained within that cell in any
form. For example, exogenous nucleic acid molecules can be
integrated into the genome of the cell or maintained in an episomal
state that can stably be passed on ("inherited") to daughter cells.
Such extra-chromosomal genetic elements (such as plasmids,
mitochondrial genome, etc.) can additionally contain selection
markers that ensure the presence of such genetic elements in
daughter cells. Moreover, the yeast cells can be stably or
transiently transformed. In addition, the yeast cells described
herein can contain a single copy, or multiple copies of a
particular exogenous nucleic acid molecule as described above.
Reduction of Enzymatic Activity
[0204] Yeast microorganisms within the scope of the invention may
have reduced enzymatic activity such as reduced PDC, GPD, ALDH, or
3-KAR activity. The term "reduced" as used herein with respect to a
particular polypeptide activity refers to a lower level of
polypeptide activity than that measured in a comparable yeast cell
of the same species. The term reduced also refers to the
elimination of polypeptide activity as compared to a comparable
yeast cell of the same species. Thus, yeast cells lacking activity
for an endogenous PDC, GPD, ALDH, or 3-KAR are considered to have
reduced activity for PDC, GPD. ALDH, or 3-KAR since most, if not
all, comparable yeast strains have at least some activity for PDC,
GPD, ALDH, or 3-KAR. Such reduced PDC, GPD, ALDH, or 3-KAR
activities can be the result of lower PDC, GPD, ALDH, or 3-KAR
concentration (e.g., via reduced expression), lower specific
activity of the PDC. GPD, ALDH, or 3-KAR, or a combination thereof.
Many different methods can be used to make yeast having reduced
PDC, GPD, ALDH, or 3-KAR activity. For example, a yeast cell can be
engineered to have a disrupted PDC-, GPD-, ALDH-, or 3-KAR-encoding
locus using common mutagenesis or knock-out technology. See, e.g.,
Methods in Yeast Genetics (1997 edition), Adams, Gottschling,
Kaiser, and Stems, Cold Spring Harbor Press (1998). In addition, a
yeast cell can be engineered to partially or completely remove the
coding sequence for a particular PDC, GPD, ALDH, or 3-KAR.
Furthermore, the promoter sequence and/or associated regulatory
elements can be mutated, disrupted, or deleted to reduce the
expression of a PDC, GPD, ALDH, or 3-KAR. Moreover, certain
point-mutation(s) can be introduced which results in a PDC, GPD,
ALDH, or 3-KAR with reduced activity. Also included within the
scope of this invention are yeast strains which when found in
nature, are substantially free of one or more PDC, GPD, ALDH, or
3-KAR activities.
[0205] Alternatively, antisense technology can be used to reduce
PDC, GPD, ALDH, or 3-KAR activity. For example, yeasts can be
engineered to contain a cDNA that encodes an antisense molecule
that prevents a PDC, GPD, ALDH, or 3-KAR from being made. The term
"antisense molecule" as used herein encompasses any nucleic acid
molecule that contains sequences that correspond to the coding
strand of an endogenous polypeptide. An antisense molecule also can
have flanking sequences (e.g., regulatory sequences). Thus
antisense molecules can be ribozymes or antisense oligonucleotides.
A ribozyme can have any general structure including, without
limitation, hairpin, hammerhead, or axhead structures, provided the
molecule cleaves RNA.
Overexpression of Heterologous Genes
[0206] Methods for overexpressing a polypeptide from a native or
heterologous nucleic acid molecule are well known. Such methods
include, without limitation, constructing a nucleic acid sequence
such that a regulatory element promotes the expression of a nucleic
acid sequence that encodes the desired polypeptide. Typically,
regulatory elements are DNA sequences that regulate the expression
of other DNA sequences at the level of transcription. Thus,
regulatory elements include, without limitation, promoters,
enhancers, and the like. For example, the exogenous genes can be
under the control of an inducible promoter or a constitutive
promoter. Moreover, methods for expressing a polypeptide from an
exogenous nucleic acid molecule in yeast are well known. For
example, nucleic acid constructs that are used for the expression
of exogenous polypeptides within Kluyveromyces and Saccharomyces
are well known (see, e.g., U.S. Pat. Nos. 4,859,596 and 4,943,529,
for Kluyveromyces and, e.g., Gellissen et al., Gene 190(1):87-97
(1997) for Saccharomyces). Yeast plasmids have a selectable marker
and an origin of replication. In addition certain plasmids may also
contain a centromeric sequence. These centromeric plasmids are
generally a single or low copy plasmid. Plasmids without a
centromeric sequence and utilizing either a 2 micron (S.
cerevisiae) or 1.6 micron (K. lactis) replication origin are high
copy plasmids. The selectable marker can be either prototrophic,
such as HIS3. TRP1, LEU2, URA3 or ADE2, or antibiotic resistance,
such as, bar, ble, hph, or kan.
[0207] In another embodiment, heterologous control elements can be
used to activate or repress expression of endogenous genes.
Additionally, when expression is to be repressed or eliminated, the
gene for the relevant enzyme, protein or RNA can be eliminated by
known deletion techniques.
[0208] As described herein, any yeast within the scope of the
disclosure can be identified by selection techniques specific to
the particular polypeptide (e.g. an isobutanol pathway enzyme)
being expressed, over-expressed or repressed. Methods of
identifying the strains with the desired phenotype are well known
to those skilled in the art. Such methods include, without
limitation, PCR, RT-PCR, and nucleic acid hybridization techniques
such as Northern and Southern analysis, altered growth capabilities
on a particular substrate or in the presence of a particular
substrate, a chemical compound, a selection agent and the like. In
some cases, immunohistochemistry and biochemical techniques can be
used to determine if a cell contains a particular nucleic acid by
detecting the expression of the encoded polypeptide. For example,
an antibody having specificity for an encoded enzyme can be used to
determine whether or not a particular yeast cell contains that
encoded enzyme. Further, biochemical techniques can be used to
determine if a cell contains a particular nucleic acid molecule
encoding an enzymatic polypeptide by detecting a product produced
as a result of the expression of the enzymatic polypeptide. For
example, transforming a cell with a vector encoding acetolactate
synthase and detecting increased acetolactate concentrations
compared to a cell without the vector indicates that the vector is
both present and that the gene product is active. Methods for
detecting specific enzymatic activities or the presence of
particular products are well known to those skilled in the art. For
example, the presence of acetolactate can be determined as
described by Hugenholtz and Starrenburg, 1992, Appl. Micro. Biot.
38:17-22.
Increase of Enzymatic Activity
[0209] Yeast microorganisms of the invention may be further
engineered to have increased activity of enzymes (e.g., increased
activity of enzymes involved in an isobutanol producing metabolic
pathway). The term "increased" as used herein with respect to a
particular enzymatic activity refers to a higher level of enzymatic
activity than that measured in a comparable yeast cell of the same
species. For example, overexpression of a specific enzyme can lead
to an increased level of activity in the cells for that enzyme.
Increased activities for enzymes involved in glycolysis or the
isobutanol pathway would result in increased productivity and yield
of isobutanol.
[0210] Methods to increase enzymatic activity are known to those
skilled in the art. Such techniques may include increasing the
expression of the enzyme by increased copy number and/or use of a
strong promoter, introduction of mutations to relieve negative
regulation of the enzyme, introduction of specific mutations to
increase specific activity and/or decrease the K.sub.M for the
substrate, or by directed evolution. See, e.g., Methods in
Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press
(2003).
Methods of Using Recombinant Microorganisms for Metabolite
Production
[0211] For a biocatalyst to produce a beneficial metabolite most
economically, it is desirable to produce said metabolite at a high
yield. Preferably, the only product produced is the desired
metabolite, as extra products (i.e. by-products) lead to a
reduction in the yield of the desired metabolite and an increase in
capital and operating costs, particularly if the extra products
have little or no value. These extra products also require
additional capital and operating costs to separate these products
from the desired metabolite.
[0212] In one aspect, the present application provides methods of
producing a desired metabolite using a recombinant described
herein. In one embodiment, the recombinant microorganism comprises
a KARI-requiring biosynthetic pathway, wherein said recombinant
microorganism comprises at least one nucleic acid molecule encoding
a KARI that is at least about 80% identical to SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28,
SEQ ID NO: 40, or SEQ ID NO: 58. In one embodiment, the KARI is
derived from the genus Shewanella. In a specific embodiment, the
KARI is derived from Shewanella sp. strain MR-4. In another
specific embodiment, the isolated nucleic acid molecule is
comprised of SEQ ID NO: 1. In another embodiment, the KARI is
derived from the genus Vibrio. In a specific embodiment, the KARI
is derived from Vibrio fischeri. In another specific embodiment,
the isolated nucleic acid molecule is comprised of SEQ ID NO: 3. In
yet another embodiment, the KARI is derived from the genus
Gramella. In a specific embodiment, the KARI is derived from
Gramella forsetii. In another specific embodiment, the isolated
nucleic acid molecule is comprised of SEQ ID NO: 5. In yet another
embodiment, the KARI is derived from the genus Cytophaga. In a
specific embodiment, the KARI is derived from Cytophaga
hutchinsonii. In another specific embodiment, the isolated nucleic
acid molecule is comprised of SEQ ID NO: 7. In yet another
embodiment, the KARI is derived from a genus selected from
Lactococcus and Streptococcus. In a specific embodiment, the KARI
is derived from Lactococcus lactis, Streptococcus equinus, or
Streptococcus infantarius. In another specific embodiment, the KARI
is encoded by SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
or SEQ ID NO: 25. In yet another embodiment, the KARI is derived
from the genus Methanococcus. In a specific embodiment, the KARI is
derived from Methanococcus maripaludis, Methanococcus vannielii, or
Methanococcus voltae. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, or SEQ ID NO: 37. In yet another embodiment, the
KARI is derived from a genus selected from Zymomonas,
Erythrobacter, Sphingomonas, Sphingobium, and Novosphingobium. In a
specific embodiment, the KARI is derived from Zymomonas mobilis,
Erythrobacter litoralis, Sphingomonas wittichii, Sphingobium
japonicum, Sphingobium chlorophenolicum, or Novosphingobium
nitrogenifigens. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:
45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO: 53.
In yet another embodiment, the KARI is derived from the genus
Bacteroides. In a specific embodiment, the KARI is derived from
Bacteroides thetaiotaomicron. In another specific embodiment, the
KARI is encoded by SEQ ID NO: 55. In yet another embodiment, the
KARI is derived from the genus Schizosaccharomyces. In a specific
embodiment, the KARI is derived from Schizosaccharomyces pombe or
Schizosaccharomyces japonicus. In another specific embodiment, the
KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61.
In yet another embodiment, the KARI has one or more modifications
or mutations at positions corresponding to amino acids selected
from: (a) alanine 71 of the Shewanella sp. KARI (SEQ ID NO: 2); (b)
arginine 76 of the Shewanella sp. KARI (SEQ ID NO: 2): (c) serine
78 of the Shewanella sp. KARI; and (d) glutamine 110 of the
Shewanella sp. KARI (SEQ ID NO: 2). In yet another embodiment, the
KARI has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) valine 48 of the L.
lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI
(SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO:
10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e)
glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f);
leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89
of the L. lactis KARI (SEQ ID NO: 10); (h) lysine 118 of the L.
lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L. lactis
KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis
KARI (SEQ ID NO: 10).
[0213] In another embodiment, the recombinant microorganism
comprises a KARI-requiring biosynthetic pathway, wherein said
recombinant microorganism comprises at least one nucleic acid
molecule encoding a KARI that is at least about 99% identical to
SEQ ID NO: 64. In one embodiment, the KARI is derived from the
genus Salmonella. In a specific embodiment, the KARI is derived
from Salmonella enterica. In another specific embodiment, the KARI
is encoded by SEQ ID NO: 63. In yet another embodiment, the KARI
has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) alanine 71 of the
S. enterica KARI (SEQ ID NO: 64); (b) arginine 76 of the S.
enterica KARI (SEQ ID NO: 64); (c) serine 78 of the S. enterica
KARI (SEQ ID NO: 64); (d) glutamine 110 of the S. enterica KARI
(SEQ ID NO: 64); (e) aspartic acid 146 of the S. enterica KARI (SEQ
ID NO: 64); (f) glycine 185 of the S. enterica KARI (SEQ ID NO:
64); and (g) lysine 433 of the S. enterica KARI (SEQ ID NO:
64).
[0214] In an exemplary embodiment, the KARI-requiring biosynthetic
pathway is a pathway for the production of a metabolite selected
from isobutanol, isoleucine, leucine, valine, pantothenate,
coenzyme A, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol,
3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, and
5-methyl-1-heptanol. In a further exemplary embodiment, the
beneficial metabolite is isobutanol.
[0215] In a method to produce a beneficial metabolite (e.g.,
isobutanol) from a carbon source, the recombinant microorganism is
cultured in an appropriate culture medium containing a carbon
source. In certain embodiments, the method further includes
isolating the beneficial metabolite (e.g., isobutanol) from the
culture medium. For example, a beneficial metabolite (e.g.,
isobutanol) may be isolated from the culture medium by any method
known to those skilled in the art, such as distillation,
pervaporation, or liquid-liquid extraction. In certain exemplary
embodiments, the beneficial metabolite is selected from isobutanol,
isoleucine, leucine, valine, pantothenate, coenzyme A, 1-butanol,
2-methyl-1-butanol, 3-methyl-1-butanol, 3-methyl-1-pentanol,
4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.
In a further exemplary embodiment, the beneficial metabolite is
isobutanol.
[0216] In one embodiment, the recombinant microorganism may produce
the beneficial metabolite (e.g., isobutanol) from a carbon source
at a yield of at least 5 percent theoretical. In another
embodiment, the microorganism may produce the beneficial metabolite
(e.g., isobutanol) from a carbon source at a yield of at least
about 10 percent, at least about 15 percent, about least about 20
percent, at least about 25 percent, at least about 30 percent, at
least about 35 percent, at least about 40 percent, at least about
45 percent, at least about 50 percent, at least about 55 percent,
at least about 60 percent, at least about 65 percent, at least
about 70 percent, at least about 75 percent, at least about 80
percent, at least about 85 percent, at least about 90 percent, at
least about 95 percent, or at least about 97.5% theoretical. In a
specific embodiment, the beneficial metabolite is isobutanol.
Distillers Dried Grains Comprising Spent Yeast Biocatalysts
[0217] In an economic fermentation process, as many of the products
of the fermentation as possible, including the co-products that
contain biocatalyst cell material, should have value. Insoluble
material produced during fermentations using grain feedstocks, like
corn, is frequently sold as protein and vitamin rich animal feed
called distillers dried grains (DDG). See, e.g., commonly owned and
co-pending U.S. Publication No. 2009/0215137, which is herein
incorporated by reference in its entirety for all purposes. As used
herein, the term "DDG" generally refers to the solids remaining
after a fermentation, usually consisting of unconsumed feedstock
solids, remaining nutrients, protein, fiber, and oil, as well as
spent yeast biocatalysts or cell debris therefrom that are
recovered by further processing from the fermentation, usually by a
solids separation step such as centrifugation.
[0218] Distillers dried grains may also include soluble residual
material from the fermentation, or syrup, and are then referred to
as "distillers dried grains and solubles" (DDGS). Use of DDG or
DDGS as animal feed is an economical use of the spent biocatalyst
following an industrial scale fermentation process.
[0219] Accordingly, in one aspect, the present invention provides
an animal feed product comprised of DDG derived from a fermentation
process for the production of a beneficial metabolite (e.g.,
isobutanol), wherein said DDG comprise a spent yeast biocatalyst of
the present invention. In an exemplary embodiment, said spent yeast
biocatalyst has been engineered to comprise at least one nucleic
acid molecule encoding a KARI that is at least about 80% identical
to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID
NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58. In one
embodiment, the KARI is derived from the genus Shewanella. In a
specific embodiment, the KARI is derived from Shewanella sp. strain
MR-4. In another specific embodiment, the isolated nucleic acid
molecule is comprised of SEQ ID NO: 1. In another embodiment, the
KARI is derived from the genus Vibrio. In a specific embodiment,
the KARI is derived from Vibrio fischeri. In another specific
embodiment, the isolated nucleic acid molecule is comprised of SEQ
ID NO: 3. In yet another embodiment, the KARI is derived from the
genus Gramella. In a specific embodiment, the KARI is derived from
Gramella forsetii. In another specific embodiment, the isolated
nucleic acid molecule is comprised of SEQ ID NO: 5. In yet another
embodiment, the KARI is derived from the genus Cytophaga. In a
specific embodiment, the KARI is derived from Cytophaga
hutchinsonii. In another specific embodiment, the isolated nucleic
acid molecule is comprised of SEQ ID NO: 7. In yet another
embodiment, the KARI is derived from a genus selected from
Lactococcus and Streptococcus. In a specific embodiment, the KARI
is derived from Lactococcus lactis, Streptococcus equinus, or
Streptococcus infantarius. In another specific embodiment, the KARI
is encoded by SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
or SEQ ID NO: 25. In yet another embodiment, the KARI is derived
from the genus Methanococcus. In a specific embodiment, the KARI is
derived from Methanococcus maripaludis, Methanococcus vannielii, or
Methanococcus voltae. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, or SEQ ID NO: 37. In yet another embodiment, the
KARI is derived from a genus selected from Zymomonas,
Erythrobacter, Sphingomonas, Sphingobium, and Novosphingobium. In a
specific embodiment, the KARI is derived from Zymomonas mobilis,
Erythrobacter litoralis, Sphingomonas wittichii, Sphingobium
japonicum, Sphingobium chlorophenolicum, or Novosphingobium
nitrogenifigens. In another specific embodiment, the KARI is
encoded by SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:
45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO: 53.
In yet another embodiment, the KARI is derived from the genus
Bacteroides. In a specific embodiment, the KARI is derived from
Bacteroides thetaiotaomicron. In another specific embodiment, the
KARI is encoded by SEQ ID NO: 55. In yet another embodiment, the
KARI is derived from the genus Schizosaccharomyces. In a specific
embodiment, the KARI is derived from Schizosaccharomyces pombe or
Schizosaccharomyces japonicus. In another specific embodiment, the
KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61.
In yet another embodiment, the KARI has one or more modifications
or mutations at positions corresponding to amino acids selected
from: (a) alanine 71 of the Shewanella sp. KARI (SEQ ID NO: 2); (b)
arginine 76 of the Shewanella sp. KARI (SEQ ID NO: 2); (c) serine
78 of the Shewanella sp. KARI; and (d) glutamine 110 of the
Shewanella sp. KARI (SEQ ID NO: 2). In yet another embodiment, the
KARI has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) valine 48 of the L.
lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI
(SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO:
10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e)
glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f);
leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89
of the L. lactis KARI (SEQ ID NO: 10); (h) lysine 118 of the L.
lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L. lactis
KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis
KARI (SEQ ID NO: 10). In another exemplary embodiment, said spent
yeast biocatalyst has been engineered to comprise at least one
nucleic acid molecule encoding a KARI that is at least about 99%
identical to SEQ ID NO: 64. In one embodiment, the KARI is derived
from the genus Salmonella. In a specific embodiment, the KARI is
derived from Salmonella enterica. In another specific embodiment,
the KARI is encoded by SEQ ID NO: 63. In yet another embodiment,
the KARI has one or more modifications or mutations at positions
corresponding to amino acids selected from: (a) alanine 71 of the
S. enterica KARI (SEQ ID NO: 64); (b) arginine 76 of the S.
enterica KARI (SEQ ID NO: 64); (c) serine 78 of the S. enterica
KARI (SEQ ID NO: 64); (d) glutamine 110 of the S. enterica KARI
(SEQ ID NO: 64); (e) aspartic acid 146 of the S. enterica KARI (SEQ
ID NO: 64); (f) glycine 185 of the S. enterica KARI (SEQ ID NO:
64); and (g) lysine 433 of the S. enterica KARI (SEQ ID NO:
64).
[0220] In certain additional embodiments, the DDG comprising a
spent yeast biocatalyst of the present invention comprise at least
one additional product selected from the group consisting of
unconsumed feedstock solids, nutrients, proteins, fibers, and
oils.
[0221] In another aspect, the present invention provides a method
for producing DDG derived from a fermentation process using a yeast
biocatalyst (e.g., a recombinant yeast microorganism of the present
invention), said method comprising: (a) cultivating said yeast
biocatalyst in a fermentation medium comprising at least one carbon
source; (b) harvesting insoluble material derived from the
fermentation process, said insoluble material comprising said yeast
biocatalyst; and (c) drying said insoluble material comprising said
yeast biocatalyst to produce the DDG.
[0222] In certain additional embodiments, the method further
comprises step (d) of adding soluble residual material from the
fermentation process to said DDG to produce DDGS. In some
embodiments, said DDGS comprise at least one additional product
selected from the group consisting of unconsumed feedstock solids,
nutrients, proteins, fibers, and oils.
[0223] This invention is further illustrated by the following
examples that should not be construed as limiting. The contents of
all references, patents, and published patent applications cited
throughout this application, as well as the Figures and the
Sequence Listing, are incorporated herein by reference for all
purposes.
Example 1
Reduction of KARI Inhibition by 2,3-Dihydroxyisovalerate (DHIV)
[0224] The purpose of this example is to show how high-performance
KARIs were identified.
Materials and Methods for Example 1
TABLE-US-00006 [0225] TABLE 2 Strain Used in Examples 1-2. GEVO3956
MATa ura3 leu2 his3 trp1 ald6::P.sub.ENO2- LI_adhA.sup.RE1
-P.sub.FBA1-Sc_TRP1 gpd1::T.sub.KI_URA3 gpd2::T.sub.KI_URA3
tma29::T.sub.KI_URA3 pdc1::P.sub.PDC1-LI_kivD2_coSc5-P.sub.FBA1-
LEU2-T.sub.LEU2-P.sub.ADH1-Bs_alsS1_coSc-T.sub.CYC1-P.sub.PGK1-
LI_kivD2_coEc-P.sub.ENO2-Sp_HIS5 pdc5::T.sub.KI_URA3
pdc6::P.sub.TDH3-Sc_AFT1-P.sub.ENO2-LI_adhA.sup.RE1-
T-.sub.KI_URA3_short-P.sub.FBA1-KI_URA3-T.sub.KI_URA3
TABLE-US-00007 TABLE 3 Plasmid Used in Example 1-2. pGV3009
P.sub.Sc_TEF1:LI_ilvD_coSc:T.sub.Sc_ADH1,
P.sub.Sc_PDC1-350:EC_ilvC_coSc.sup.P2D1_A1_his6,
P.sub.Sc_TPI1:G418.sup.R,P.sub.Sc_ENO2:LI_adhA.sup.RE1, CEN/ARS
origin of replication, Ap.sup.R, pMB1 origin of replication
[0226] In this example, a series of KARI genes were individually
expressed from a yeast promoter in conjunction with other
components of an isobutanol production pathway in yeast such that
KARI was the limiting enzyme in the pathway and the amount of
isobutanol produced during a fermentation was dependent on the KARI
activity level. In this system, the S. cerevisiae host strain
GEV03956, which expresses ALS and KIVD enzymes, was used to produce
isobutanol when supplied with a low copy number plasmid expressing
KARI, DHAD, and ADH enzymes.
[0227] KARIs were identified and grouped by bioinformatic and
phylogenetic methods based on the amino acid sequence. Individual
KARIs were chosen for the above analysis to provide a
representative sample of broadly diverse clades. KARI genes were
designed and synthesized based on the primary amino acid sequence
of the chosen KARI, with codon optimization of the genes for
expression in S. cerevisiae. These genes were cloned downstream of
the Sc_PDC1.sup.-350 promoter in pGV3009 to replace the
Ec_IlvC_coSc.sup.P2D1-A1.sup.--.sup.his6 gene present in the
plasmid.
[0228] Shake Flask Fermentations:
[0229] Shake flask fermentations using GEV03956 carrying these
individual plasmids were performed together in experiments with
GEV03956 carrying pGV3022 (derived from pGV3009 but containing the
E. coli ilvC_coSc gene expressed from the Sc_PDC1.sup.-350
promoter) and GEV03956 carrying pGV3012 (equivalent to pGV3009
lacking the Sc_PDC1.sup.-350 promoter and KARI gene) for comparison
of isobutanol production. The shake flask fermentations were
performed as follows. The strains were grown overnight in 3 mL of
YPD medium containing 1% v/v ethanol and 0.1 g/L G418 at 30.degree.
C. and 250 rpm. The OD.sub.600 of these cultures was determined
after overnight growth and the appropriate amount of culture was
added to 50 mL of YP medium containing 5% w/v glucose, 1% v/v
ethanol, 200 mM MES, pH 6.5, and 0.1 g/L G418 to obtain an
OD.sub.600 of 0.1 in 250 mL baffled flasks with sleeve caps.
Cultures were incubated at 30.degree. C. and 250 rpm overnight. The
OD.sub.600 of these cultures was determined after overnight growth
and the appropriate amount of culture to total 250 ODs was added to
50 mL Falcon tubes and centrifuged at 2700.times.g for 5 minutes.
The supernatant was removed and cells were resuspended in 50 mL of
YP medium containing 8% w/v glucose, 1% v/v ethanol, 200 mM MES, pH
6.5, and 0.1 g/L G418 to obtain a final OD.sub.600 of 5 OD per ml.
At t=0 the OD.sub.600 of each flask was determined. The
fermentation cultures were incubated at 30.degree. C. and 250 rpm
in non-baffled 250 mL flasks with vented screw cap tops. After 24,
48 and 72 hours of incubation, 1.5 mL of culture was removed into
1.5 mL microcentrifuge tubes from each culture. OD.sub.600 values
were determined from the samples and the remainder of each sample
was centrifuged for 10 min at 14,000 rpm in a microcentrifuge and 1
mL of the supernatant was removed to be submitted for gas
chromatographic analysis. Analysis of volatile organic compounds,
including ethanol and isobutanol, was performed on an Agilent 6890
gas chromatograph (GC) fitted with a 7683B liquid autosampler, a
split/splitless injector port, a ZB-FFAP column (Phenomenex 30 m
length, 0.32 mm ID, 0.25 .mu.M film thickness) connected to a flame
ionization detector (FID). The temperature program is as follows:
230.degree. C. for the injector, 300.degree. C. for the detector,
100.degree. C. oven for 1 minute, 35.degree. C./minute gradient to
230.degree. C., and then hold for 2.5 min. Analysis is performed
using authentic standards (>98%, obtained from Sigma-Aldrich),
and a 6-point calibration curve with 1-pentanol as the internal
standard. Injection size is 0.5 .mu.L with a 50:1 split and run
time is 7.4 min.
[0230] Results:
[0231] KARI gene clones resulting in isobutanol production
equivalent to or higher than fermentations with pGV3022 or within
two standard deviations below that of fermentations with pGV3022,
averaged from multiple experiments, were chosen as encoding
high-performing KARIs. These fermentations identified the following
as high-performance KARIs: Shewanella sp. strain MR-4 (SEQ ID NO:
2), Vibrio fischeri strain ES114 (SEQ ID NO: 4), Gramella forsetii
strain KT0803 (SEQ ID NO: 6), and Cytophaga hutchinsonii strain
ATCC33406 (SEQ ID NO: 8). Table 4 shows the results of 48 hr and 72
hr isobutanol fermentation timepoints.
TABLE-US-00008 TABLE 4 Isobutanol Titers from Fermentations with
High-Performance KARIs as compared to E. coli KARI. KARI Gene 48 h
isobutanol 72 h isobutanol Expressed Titer (g/L) Titer (g/L)
Shewanella sp. MR-4 4.01 .+-. 0.61 4.60 .+-. 0.53 Vibrio fischeri
4.27 .+-. 0.27 4.52 .+-. 0.30 Gramella forsetii 4.38 .+-. 0.69 4.45
.+-. 0.50 Cytophaga hutchinsonii 3.49 .+-. 0.20 4.26 .+-. 0.31 E.
coli 3.93 .+-. 1.16 4.72 .+-. 0.90 (Mean of 3 experiments .+-. 2
standard deviations)
[0232] Each of these identified KARIs share the property of being
long-form KARIs. Long-form KARIs are found in plants, algae, and
some bacteria, while short-form KARIs are found in fungi and
bacteria. The amino acid sequences of these high-performing
bacterial long form KARIs were aligned with the sequences of 103
other KARIs representing broad biological diversity of KARIs chosen
from the bioinformatic and phylogenetic analysis above and were
used to generate a phylogenetic tree in Clone Manager using the
"Align Multiple Sequences" feature to perform a Multi-Way alignment
of the amino acid sequences using the BLOSUM62 scoring matrix and
otherwise default parameters. This analysis identified the
Shewanella sp., Vibrio fischeri, Gramella forsetii, Cytophaga
hutchinsonii, and E. coli KARIs as all belonging to a distinct
clade of closely related bacterial long-form KARIs (see FIG. 2 of
U.S. Provisional Application No. 506,562, which is herein
incorporated by reference). A separate clade of bacterial long form
KARIs contained KARIs from the symbiotic bacteria Buchnera
aphidicola and Candidatus blochmannia species.
[0233] The Shewanella sp. KARI sequence amino acid sequence (SEQ ID
NO: 2) was used to identify the 500 closest protein sequences in
the GenBank database using the blastp algorithm on the
non-redundant protein sequence database using the default
parameters. The COBALT Multiple Alignment link from the BLAST
results page was used to perform a multiple alignment of the 500
closest protein sequences plus the Shewanella sp. KARI sequence
(SEQ ID NO: 2) using the default parameters. This alignment was
downloaded as a "Fasta plus gaps" file and opened with the Clone
Manager "Align Multiple Sequences" feature to perform a Multi-Way
alignment of the amino acid sequences using the BLOSUM62 scoring
matrix and otherwise default parameters to generate a phylogenetic
tree of the sequences.
[0234] KARI sequences representing the major clades of this tree
were chosen and used to generate a representative subset
phylogenetic tree. The resulting subset phylogenetic tree showed a
clade of proteins sequences containing the Shewanella sp., Vibrio
fischeri, Gramella forsetii, Cytophaga hutchinsonii, and E. coli
KARIs and KARIs that were closely related to those sequences (see
FIG. 3 of U.S. Provisional Application No. 506,562, which is herein
incorporated by reference). Each of the Shewanella sp., Vibrio
fischeri, Gramella forsetii, Cytophaga hutchinsonii, and E. coli
KARIs were in a separate subclade in this tree, indicating that
high-performing KARIs for isobutanol production in yeast can be
found throughout this overall clade of long-form bacterial
KARIs.
Example 2
[0235] The purpose of this example is to show how additional
high-performance KARIs were identified.
[0236] In this example, a series of KARI genes were individually
expressed from a yeast promoter in conjunction with other
components of an isobutanol production pathway in yeast such that
KARI was the limiting enzyme in the pathway and the amount of
isobutanol produced during a fermentation was dependent on the KARI
activity level. In this system, the S. cerevisiae host strain
GEV03956, which expresses ALS and KIVD enzymes, was used to produce
isobutanol when supplied with a low copy number plasmid expressing
KARI, DHAD, and ADH enzymes.
[0237] KARIs were identified and grouped by bioinformatic and
phylogenetic methods based on the amino acid sequence. Individual
KARIs were chosen for the above analysis to provide a
representative sample of broadly diverse clades. KARI genes were
designed and synthesized based on the primary amino acid sequence
of the chosen KARI, with codon optimization of the genes for
expression in S. cerevisiae. These genes were cloned downstream of
the Sc_PDC1.sup.-350 promoter in pGV3009 to replace the
Ec_IlvC_coSc.sup.P2D-A1.sup.--.sup.his6 gene present in the
plasmid.
[0238] Shake Flask Fermentations:
[0239] Shake flask fermentations using GEV03956 carrying these
individual plasmids were performed together in experiments with
GEV03956 carrying pGV3022 (derived from pGV3009 but containing the
E. coli ilvC_coSc gene expressed from the Sc_PDC1.sup.-350
promoter) and GEV03956 carrying pGV3012 (equivalent to pGV3009
lacking the Sc_PDC1.sup.-350 promoter and KARI gene) for comparison
of isobutanol production. The shake flask fermentations were
performed as follows. The strains were grown overnight in 3 mL of
YPD medium containing 1% v/v ethanol and 0.1 g/L G418 at 30.degree.
C. and 250 rpm. The OD.sub.600 of these cultures was determined
after overnight growth and the appropriate amount of culture was
added to 50 mL of YP medium containing 5% w/v glucose, 1% v/v
ethanol, 200 mM MES, pH 6.5, and 0.1 g/L G418 to obtain an
OD.sub.600 of 0.1 in 250 mL baffled flasks with sleeve caps.
Cultures were incubated at 30.degree. C. and 250 rpm overnight. The
OD.sub.600 of these cultures was determined after overnight growth
and the appropriate amount of culture to total 250 ODs was added to
50 mL Falcon tubes and centrifuged at 2700.times.g for 5 minutes.
The supernatant was removed and cells were resuspended in 50 mL of
YP medium containing 8% w/v glucose, 1% v/v ethanol, 200 mM MES, pH
6.5, and 0.1 g/L G418 to obtain a final OD.sub.600 of 5 OD per ml.
At t=0 the OD.sub.600 of each flask was determined. The
fermentation cultures were incubated at 30.degree. C. and 250 rpm
in non-baffled 250 mL flasks with vented screw cap tops. After 24,
48 and 72 hours of incubation, 1.5 mL of culture was removed into
1.5 mL microcentrifuge tubes from each culture. OD.sub.600 values
were determined from the samples and the remainder of each sample
was centrifuged for 10 min at 14,000 rpm in a microcentrifuge and 1
mL of the supernatant was removed to be submitted for gas
chromatographic analysis. Analysis of volatile organic compounds,
including ethanol and isobutanol, was performed on an Agilent 6890
gas chromatograph (GC) fitted with a 7683B liquid autosampler, a
split/splitless injector port, a ZB-FFAP column (Phenomenex 30 m
length, 0.32 mm ID, 0.25 .mu.M film thickness) connected to a flame
ionization detector (FID). The temperature program is as follows:
230.degree. C. for the injector, 300.degree. C. for the detector,
100.degree. C. oven for 1 minute, 35.degree. C./minute gradient to
230.degree. C., and then hold for 2.5 min. Analysis is performed
using authentic standards (>98%, obtained from Sigma-Aldrich),
and a 6-point calibration curve with 1-pentanol as the internal
standard. Injection size is 0.5 .mu.L with a 50:1 split and run
time is 7.4 min.
[0240] Results:
[0241] KARI gene clones resulting in isobutanol production
equivalent to or higher than fermentations with pGV3022 or within
two standard deviations below that of fermentations with pGV3022,
averaged from multiple experiments, were chosen as encoding
high-performing KARIs. These fermentations identified the following
as high-performance KARIs: Lactococcus lactis strain KF147 (SEQ ID
NO: 10), Methanococcus maripaludis strain C5 (SEQ ID NO: 28),
Zymomonas mobilis strains ZM4, ATCC10988, and NCIMB 11163 (SEQ ID
NO: 40), Bacteroides thetaiotaomicron strain VPI-5482 (SEQ ID NO:
56), and Schizosaccharomyces pombe strain ATCC33406 (SEQ ID NO:
58). Table 5 shows the results of 48 hr and 72 hr isobutanol
fermentation timepoints.
TABLE-US-00009 TABLE 5 Isobutanol Titers from Fermentations with
High-Performance KARIs as compared to E. coli KARI. KARI Gene 48 h
Isobutanol 72 h Isobutanol Expressed Titer (g/L) Titer (g/L)
Lactococcus lactis 5.68 .+-. 0.90 6.21 .+-. 0.62 Methanococcus
maripaludis 4.03 .+-. 0.18 4.70 .+-. 0.18 Zymomonas mobilis 4.00
.+-. 0.32 4.67 .+-. 0.24 Bacteroides thetaotaomicron 3.21 .+-. 0.03
4.07 .+-. 0.01 Schizosaccharomyces pombe 3.33 .+-. 0.30 3.89 .+-.
0.23 (.DELTA.54tr)* E. coli 3.93 .+-. 1.16 4.72 .+-. 0.90 (Mean of
3 experiments .+-. 2 standard deviations) *Truncated by removal of
first 54 AA encoding MTS (SEQ ID NO: 90).
[0242] Each of these identified KARIs share the property of being
short-form KARIs. Short-form KARIs are found in fungi and bacteria,
while long-form KARIs are found in plants, algae, and some
bacteria. An additional 21 short-form KARIs tested did not meet the
yeast isobutanol fermentation criteria in the above
experiments.
[0243] The Lactococcus lactis, Methanococcus maripaludis, and
Zymomonas mobilis KARIs were also identified as performing as well
or better than the E. coli KARI in shake flask fermentations when
expressed from a high copy number plasmid. Genes encoding these
KARIs were cloned downstream of a Sc_TDH3 promoter to replace the
Ec_ilvC_coSc.sup.P2D1-A1.sup.--.sup.his6 gene present in that
plasmid.
[0244] Shake flask fermentations of GEVO3956 carrying these
individual plasmids were performed together with GEVO3956 carrying
pGV2911 (derived from pGV2901 but containing the E. coli ilvC_coSc
gene expressed from the Sc_TDH3 promoter) for comparison of
isobutanol production. The shake flask fermentations were performed
as follows. The strains were grown overnight in 3 mL of YPD medium
containing 1% v/v ethanol and 0.1 g/L G418 at 30.degree. C. and 250
rpm. The OD.sub.600 of these cultures was determined after
overnight growth and the appropriate amount of culture was added to
50 mL of YP medium containing 5% w/v glucose, 1% v/v ethanol, 200
mM MES, pH 6.5, and 0.1 g/L G418 to obtain an OD.sub.600 of 0.1 in
250 mL baffled flasks with sleeve caps. Cultures were incubated at
30.degree. C. and 250 rpm overnight. The OD.sub.600 of these
cultures was determined after overnight growth and the appropriate
amount of culture to total 250 ODs was added to 50 mL Falcon tubes
and centrifuged at 2700.times.g for 5 minutes. The supernatant was
removed and cells were resuspended in 50 mL of YP medium containing
8% w/v glucose, 1% v/v of a stock of 3 g/L ergosterol and 132 g/L
Tween 80 dissolved in ethanol, 200 mM MES, pH 6.5, and 0.2 g/L G418
to obtain a final OD.sub.600 of 5 OD per ml. At t=0 the OD.sub.600
of each flask was determined. The fermentation cultures were
incubated at 30.degree. C. and 250 rpm in non-baffled 250 mL flasks
with vented screw cap tops. After 24, 46-48 and 72 hours of
incubation, 1.5 mL of culture was removed into 1.5 mL
microcentrifuge tubes from each culture. OD.sub.600 values were
determined from the samples and the remainder of each sample was
centrifuged for 10 min at 14,000 rpm in a microcentrifuge and 1 mL
of the supernatant was removed to be submitted for gas
chromatographic analysis. Analysis of volatile organic compounds,
including ethanol and isobutanol, was performed on an Agilent 6890
gas chromatograph (GC) fitted with a 7683B liquid autosampler, a
split/splitless injector port, a ZB-FFAP column (Phenomenex 30 m
length, 0.32 mm ID, 0.25 .mu.M film thickness) connected to a flame
ionization detector (FID). The temperature program is as follows:
230.degree. C. for the injector, 300.degree. C. for the detector,
100.degree. C. oven for 1 minute, 35.degree. C./minute gradient to
230.degree. C., and then hold for 2.5 min. Analysis is performed
using authentic standards (>98%, obtained from Sigma-Aldrich),
and a 6-point calibration curve with 1-pentanol as the internal
standard. Injection size is 0.5 .mu.L with a 50:1 split and run
time is 7.4 min.
[0245] The M. maripaludis, Z. mobilis, and L. lactis KARIs,
expressed from plasmids in GEV03956, resulted in isobutanol titers
within two standard deviations of that produced from pGV2911 in
GEV03956 at both the 48 hour and 72 hour fermentation time points
(Tables 6 and 7).
TABLE-US-00010 TABLE 6 Isobutanol titers from isobutanol
fermentations with the M. maripaludis and Z. mobilis KARIs versus
the E. coli KARI expressed from high copy number plasmids. KARI
Gene 48 h Isobutanol 72 h Isobutanol Expressed Titer (g/L) Titer
(g/L) M. maripaludis 11.26 .+-. 0.84 12.60 .+-. 1.08 Z. mobilis
11.47 .+-. 0.07 13.13 .+-. 0.02 E. coli 12.34 .+-. 1.06 13.61 .+-.
1.06 (Mean of 3 experiments .+-. 2 standard deviations)
TABLE-US-00011 TABLE 7 Isobutanol titers from isobutanol
fermentations with the L. lactis KARI versus the E. coli KARI
expressed from high copy number plasmids. KARI Gene 48 h Isobutanol
72 h Isobutanol Expressed Titer (g/L) Titer (g/L) L. lactis 7.25
.+-. 0.88 14.51 .+-. 2.16 E. coli 7.39 .+-. 2.64 13.54 .+-. 4.54
(Mean of 3 experiments .+-. 2 standard deviations)
[0246] The L. lactis, M. maripaludis, Z. mobilis, B.
thetaiotaomicron, and S. pombe KARI amino acid sequences were used
to identify the closest protein sequences in the GenBank database
using the blastp algorithm on the non-redundant protein sequence
database using the default parameters. Table 8 discloses KARI
sequences that have .gtoreq.80% amino acid identity with the L.
lactis, M. maripaludis, Z. mobilis, or S. pombe KARIs.
TABLE-US-00012 TABLE 8 KARI sequences having .gtoreq. 80% amino
acid identity with the L. lactis, M. maripaludis, Z. mobilis, or S.
pombe KARIs. Reference Sequence Origin SEQ ID NO: L. lactis subsp.
lactis L. lactis subsp. lactis II1430 12 KF147 L. lactis subsp.
lactis CV56 14 (SEQ ID NO: 10) L. lactis subsp. cremoris MG1363 16
L. lactis subsp. cremoris NZ9000 18 L. lactis subsp. cremoris SK11
20 L. lactis subsp. lactis NCDO2118 22 S. equinus ATCC 9812 24 S.
infantarius subsp. infantarius 26 ATCC BAA-102 M. maripaludis M.
maripaludis C7 30 strain C5 M. maripaludis C6 32 (SEQ ID NO: 28) M.
maripaludis S2 34 M. vannielii SB 36 M. voltae A3 38 Z. mobilis
strain Erythrobacter sp. NAP1 42 ZM4 Sphingomonas wittichii RW1 44
(SEQ ID NO: 40) Sphingobium japonicum UT26S 46 Erythrobacter
litoralis HTCC2594 48 Sphingobium chlorophenolicum L-1 50
Sphingomonas sp. S17 52 Novosphingobium nitrogenifigens 54 DSM
19370 S. pombe strain S. pombe PR745 60 972 h-* (Fuii Length: S.
japonicus yFS275 62 SEQ ID NO: 58)
[0247] A phylogenetic tree that discloses the database
identification numbers of 50 KARI sequences that have 278% amino
acid identity with the B. thetaiotaomicron KARI and their
phylogenetic relationship with the B. thetaiotaomicron KARI was
generated (see FIG. 2 of U.S. Provisional Application No. 506,564,
which is herein incorporated by reference).
[0248] The alignments of the B. thetaiotaomicron KARI protein with
42 closely related KARIs (85-97% amino acid sequence identity in
aligned regions) from other Bacteroides strains and species
indicates that the N-terminal 12 amino acids of the B.
thetaiotaomicron KARI are not conserved and are missing from these
related proteins. There is no clearly identifiable ribosome binding
site with appropriate spacing upstream of either the annotated
start codon of the B. thetaiotaomicron KARI gene sequence annotated
from the B. thetaiotaomicron genome sequence project (NCBI
reference sequence NC.sub.--004663) or the methionine codon for
amino acid position 13 of the annotated protein (nucleotide
positions 2600124-2600122 from NCBI reference sequence
NC.sub.--004663). As such it is difficult to determine whether the
start codon in B. thetaiotaomicron is at the annotated position
(nucleotides 2600160-2600158 from NCBI reference sequence
NC.sub.--004663) or at the methionine codon for amino acid position
13 of the annotated protein (nucleotide positions 2600124-2600122
from NCBI reference sequence NC.sub.--004663). Based on these
analyses, a version of the B. thetaiotaomicron KARI lacking the
N-terminal 12 amino acids of the annotated protein may function as
well or better for isobutanol production in yeast compared with
performance of the B. thetaiotaomicron KARI. Such a protein would
have the sequence of SEQ ID NO: 88.
Example 3
Cofactor Switch of the L. lactis KARI
[0249] The purpose of this example is to demonstrate how the
cofactor specificity of the L. lactis KARI can be switched from
NADPH to NADH.
[0250] Similar to all known native KARI enzymes, the L. lactis KARI
is NADPH-dependent. To enable the enzyme's use in the production of
isobutanol at theoretical yield and/or under anaerobic conditions,
the enzyme's cofactor usage was switched from NADPH to NADH.
Materials and Methods for Example 3
TABLE-US-00013 [0251] TABLE 9 Strains Used in Example 3. Strain
Genotype/Source E. coli F.sup.- ompT gal dcm lon
hsdS.sub.B(r.sub.B.sup.-m.sub.B.sup.-) .lamda. (DE3 BL21 (DE3)
[lacI lacUV5-T7 gene 1 ind1 sam7 nin5]
TABLE-US-00014 TABLE 10 Plasmids Used in Example 3. Plasmid
Genotype pET22b(+) PT7, bla, ori pBR322, lacI, C-term 6xHis
pET[ilvC] PT7::Ec_ilvC_coEc.sup.his6, bla, oripBR322, lacI pGV3281
PT7::LI_KARI_coSc.sup.his6, bla, oripBR322, lacI pETLI1A9
PT7::LI_KARI.sup.1A9_coSc.sup.his6, bla, opripBR322, lacI pETLI1G2
PT7::LI_KARI.sup.1G2_coSc.sup.his6, bla, oripBR322, lacI pETLI1C2
PT7::LI_KARI.sup.1C2_coSc.sup.his6, bla, oripBR322, lacI pETLI1G5
PT7::LI_KARI.sup.1G5_coSc.sup.his6, bla, oripBR322, lacI pETLI4H8
PT7::LI_KARI.sup.4H8_coSc.sup.his6, bla, oripBR322, lacI pETLI3C7
PT7::LI_KARI.sup.3C7_coSc.sup.his6, bla, oripBR322, lacI
pETLINKRGen6a PT7::LI_NKR.sup.Gen6a_coSc.sup.his6, bla, oripBR322,
lacI pETLINKRGen6b PT7::LI_NKR.sup.Gen6b_coSc.sup.his6, bla,
oripBR322, lacI
TABLE-US-00015 TABLE 11 Primers Used in Example 3. # Primer name
Sequence 1 T7_for TAATACGACTCACTATAGGG (SEQ ID NO: 91) 2 T7_rev
GCTAGTTATTGCTCAGCGG (SEQ ID NO: 92) 3 LIKARI_Y26NNK_for
ATCGCCGTTATTGGANNKGG TTCACAAGGACATGCCCATG (SEQ ID NO: 93) 4
LIKARI_Y26NNK_rev CATGGGCATGTCCTTGTGAA CCMNNTCCAATAACGGCGAT (SEQ ID
NO: 94) 5 LIKARI_V48NNK_for CAATGTTATCATTGGTNNKA
GGCACGGAAAATCTTTTGAT (SEQ ID NO: 95) 6 LIKARI_V48NNK_rev
ATCAAAAGATTTTCCGTGCC TMNNACCAATGATAACATTG (SEQ ID NO: 96) 7
LIKARI_R49NNK_for GTTATCATTGGTGTANNKCA CGGAAAATCTTTTG (SEQ ID NO:
97) 8 LIKARI_R49NNK_rev CAAAAGATTTTCCGTGMNNT ACACCAATGATAAC (SEQ ID
NO: 98) 9 LIKARI_G51NNK_for ATTGGTGTAAGGCACNNKAA
ATCTTTTGATAAAGCTAAG (SEQ ID NO: 99) 10 LIKARI_G51NNK_rev
CTTAGCTTTATCAAAAGATTT MNNGTGCCTTACACCAAT (SEQ ID NO: 100) 11
LIKARI_K52NNK_for GGTGTAAGGCACGGANNKTC TTTTGATAAAGCTAAGGAA (SEQ ID
NO: 101) 12 LIKARI_K52NNK_rev TTCCTTAGCTTTATCAAAAG
AMNNTCCGTGCCTTACACC (SEQ ID NO: 102) 13 LIKARI_S53NNK_for
GTGTAAGGCACGGAAAANNK TTTGATAAAGCTAAGGA (SEQ ID NO: 103) 14
LIKARI_S53NNK_rev TCCTTAGCTTTATCAAAMNN TTTTCCGTGCCTTACAC (SEQ ID
NO: 104) 15 LIKARI_L85NNK_for TTTGGCACCAGATGAGNNKC
AACAATCCATATACGAG (SEQ ID NO: 105) 16 LIKARI_L85NNK_rev
CTCGTATATGGATTGTTGMN NCTCATCTGGTGCCAM (SEQ ID NO: 106) 17
LIKARI_I89NNK_for GAGTTGCAACAATCCNNKTA CGAGGAGGATATCAAGCCT (SEQ ID
NO: 107) 18 LIKARI_I89NNK_rev AGGCTTGATATCCTCCTCGT
AMNNGGATTGTTGCAACTC (SEQ ID NO: 108) 19 LI_recomb_1a_for
GGGCACAATGTTATCATTGG TSYACBACACGGAMWATCTT TTGATAAAGCTAAGGAAG (SEQ
ID NO: 109) 20 LI_recomb_1b_for GGGCACAATGTTATCATTGG
TSYAGTGCACGGAMWATCTT TTGATAAAGCTAAGGAAG (SEQ ID NO: 110) 21
LI_recomb_1c_for GGGCACAATGTTATCATTGG TSYATCGCACGGAMWATCTT
TTGATAAAGCTAAGGAAG (SEQ ID NO: 111) 22 LI_recomb_1a_rev
CTTCCTTAGCTTTATCAAAA GATWKTCCGTGTVGTRSAC CAATGATAACATTGTGCCC (SEQ
ID NO: 112) 23 LI_recomb_1b_rev CTTCCTTAGCTTTATCAAAA
GATWKTCCGTGCACTRSACC AATGATAACATTGTGCCC (SEQ ID NO: 113) 24
LI_recomb_1c_rev CTTCCTTAGCTTTATCAAAA GATWKTCCGTGCGATRSACC
AATGATAACATTGTGCCC (SEQ ID NO: 114) 25 LI_recomb_2a_for
GGCACCAGATGAGRCACAAC AATCCATATACGAGGAGGAT ATCAAGCC (SEQ ID NO: 115)
26 LI_recomb_2b_for GGCACCAGATGAGRCACAAC AATCCGCATACGAGGAGGAT
ATCAAGCC (SEQ ID NO: 116) 27 LI_recomb_2c_for GGCACCAGATGAGTTGCAAC
AATCCATATACGAGGAGGAT ATCAAGCC (SEQ ID NO: 117) 28 LI_recomb_2d_for
GGCACCAGATGAGTTGCAAC AATCCGCATACGAGGAGGAT ATCAAGCC (SEQ ID NO: 118)
29 LI_recornb_2a_rev GGCTTGATATCCTCCTCGTA TATGGATTGTTGTGYCTCAT
CTGGTGCC (SEQ ID NO: 119) 30 LI_recomb_2b_rev GGCTTGATATCCTCCTCGTA
TGCGGATTGTTGTGYCTCAT CTGGTGCC (SEQ ID NO: 120) 31 LI_recomb_2c_rev
GGCTTGATATCCTCCTCGTA TATGGATTGTTGCAACTCAT CTGGTGCC (SEQ ID NO: 121)
32 LI_recomb_2d_rev GGCTTGATATCCTCCTCGTA TGCGGATTGTTGCAACTCAT
CTGGTGCC (SEQ ID NO: 122) 33 LI_recomb_3KS_for CACGGAAAATCTTTTGATAA
AGCTAAGGAA (SEQ ID NO: 123) 34 LI_recomb_3LS_for
CACGGACTATCTTTTGATAA AGCTAAGGAA (SEQ ID NO: 124) 35
LI_recomb_3KD_for CACGGAAAAGATTTTGATAA AGCTAAGGAA (SEQ ID NO: 125)
36 LI_recomb_3LD_for CACGGACTAGATTTTGATAA AGCTAAGGAA (SEQ ID NO:
126) 37 LI_recomb_3KS_rev TTCCTTAGCTTTATCAAAAG ATTTTCCGTG (SEQ ID
NO: 127) 38 LI_recomb_3LS_rev TTCCTTAGCTTTATCAAAAG ATAGTCCGTG (SEQ
ID NO: 128) 39 LI_recomb_3KD_rev TTCCTTAGCTTTATCAAAAT CTTTTCCGTG
(SEQ ID NO: 129) 40 LI_recomb_3LD_rev TTCCTTAGCTTTATCAAAAT
CTAGTCCGTG (SEQ ID NO: 130) 41 LI_K52NNkS53NNK_for
GGTCTACCACACGGANNKNN KTTTGATAAAGCTAAG (SEQ ID NO: 131) 42
LI_K52NNkS53NNK_rev CTTAGCTTTATCAAAMNNMN NTCCGTGTGGTAGACC (SEQ ID
NO: 132) 43 E59K_recomb_rev AAAAGTTTCGAATCCATCTT YCTTAGCTTTATC (SEQ
ID NO: 133) 44 E59K_recomb_for GATAAAGCTAAGRAAGATGG ATTCGAAACTTTT
(SEQ ID NO: 134) 45 A70V_recomb_rev ATCTGCCTTAGCTACTRCTT
CACCTACTTCAAA (SEQ ID NO: 135) 46 A70V_recomb_for
TTTGAAGTAGGTGAAGYAGT AGCTAAGGCAGAT (SEQ ID NO: 136) 47
K118E/D122G_recomb_for GGATACATCRAAGTCCCAGA GGRCGTGGACGTGTTTATG
(SEQ ID NO: 137) 48 K118E/D122G_recomb_rev CATAAACACGTCCACGYCCT
CTGGGACTTYGATGTATCC (SEQ ID NO: 138) 49 H135L_recomb_rev
GGTCCTTCTAACAAGGWGGC CTGGTGCTTTTGG (SEQ ID NO: 139) 50
H135L_recomb_for CCAAAAGCACCAGGCCWCCT TGTTAGAAGGACC (SEQ ID NO:
140) 51 T182S_recomb_rev CTCTTCCTTGAAAGTGSTTT CAATGATGCCGAC (SEQ ID
NO: 141) 52 T182S_recomb_for GTCGGCATCATTGAAASCAC TTTCAAGGAAGAG
(SEQ ID NO: 142) 53 E320K_recomb_rev CATAGCTTGTCTAAGTTYTG
CCCCTATCTTTTC (SEQ ID NO: 143) 54 E320K_recomb_for
GAAAAGATAGGGGCARAACT TAGACAAGCTATG (SEQ ID NO: 144) * A (Adenine),
G (Guanine), C (Cytosine), T (Thymine), R (Purine - A or G), Y
(Pyrimidine - C or T), N (Any nucleotide), S (Strong - G or C), M
(Amino - A or C), K (Keto - G or T), B (Not A - C, G, or T), W
(Weak - A or T), V (Not T - A, C, or G)
[0252] Heterologous Expression of Wild-Type L. lactis KARI in E.
coli:
[0253] Expression of wild-type L. lactis KARI was conducted in a
2-L baffled Erlenmeyer flask filled with 1 L LB.sub.amp (Luria
Bertani Broth, Research Products International Corp, supplemented
with 100 .mu.g/mL ampicillin) inoculated with overnight culture to
an initial OD.sub.600 of 0.1. After growing the expression culture
at 37.degree. C. with shaking at 250 rpm for 4 h, the cultivation
temperature was dropped to 25.degree. C., and KARI expression was
induced with IPTG to a final concentration of 0.5 mM. After 24 h at
25.degree. C. and shaking at 250 rpm, the cells were pelleted at
5,300 g for 10 min and then frozen at -20.degree. C. until further
use.
[0254] Heterologous Expression of L. lactis KARI Variants in E.
coli:
[0255] The expression of L. lactis KARI variants was conducted in
0.25-L Erlenmeyer flasks filled with 50 mL LB.sub.amp (Luria
Bertani Broth, Research Products International Corp, supplemented
with 100 .mu.g/mL ampicillin) inoculated with overnight culture to
an initial OD.sub.600 of 0.1. After growing the expression cultures
at 37.degree. C. with shaking at 250 rpm for 4 h, the cultivation
temperature was dropped to 25.degree. C., and KARI expression was
induced with IPTG to a final concentration of 0.5 mM. After 24 h at
25.degree. C. and shaking at 250 rpm, the cells were pelleted at
5,300 g for 10 min and then frozen at -20.degree. C. until further
use.
[0256] Histrap Purification of L. lactis KARI: L. lactis KARI was
purified over a 5-mL histrap column.
[0257] Histrap Purification of L. lactis KARI Variants:
[0258] L. lactis KARI variants were purified over 1-mL histrap
columns.
[0259] Preparation of Enantiopure (S)-2-Acetolactate:
[0260] Enzymatic synthesis of (S)-2-acetolactate was performed in
an anaerobic flask. The reaction was carried out in a total volume
of 55 mL containing 20 mM potassium phosphate buffer, pH 7.0, 1 mM
MgCl.sub.2, 0.05 mM thiamine pyrophosphate (TPP), and 200 mM sodium
pyruvate. The synthesis was initiated by the addition of 65 units
of purified B. subtilis acetolactate synthase (Bs_AlsS), and the
reaction was incubated at 30.degree. C. (in a static incubator) for
7.5 hours. A buffer exchange was performed on the purified Bs_AlsS
before the synthesis to remove as much glycerol as possible. This
was done using a microcon filter with a 50 kDa nominal molecular
weight cutoff membrane to filter 0.5 mL of the purified enzyme
until only 50 .mu.L were left on top of the membrane. 450 .mu.L of
20 mM KPO.sub.4 pH 7.0, 1 mM MgCl.sub.2, and 0.05 mM TPP were then
added to the membrane and filtered again; this process was repeated
three times. The final acetolactate concentration was determined by
liquid chromatography and was .about.200 mM.
[0261] KARI Assay in 1-mL Scale to Measure NADPH and NADH K.sub.M
Values:
[0262] L. lactis KARI activity or activities of its variants were
assayed kinetically by monitoring the decrease in NADPH or NADH
concentration by measuring the change in absorbance at 340 nm. An
assay buffer was prepared containing 100 mM potassium phosphate pH
7.0, 1 mM DTT. 2.5 mM (S)-2-acetolactate, and 10 mM MgCl.sub.2
(final concentrations in the 1-mL assay, accounting for dilution
with enzyme and cofactor). Fifty .mu.L purified enzyme and 930
.mu.L of the assay buffer were placed into a 1-mL cuvette. The
reaction was initiated by addition of 20 .mu.L NADPH or NADH (200
.mu.M final concentration) for a general activity assay.
Michaelis-Menten constants of the cofactors were determined with
varying concentrations of NADPH (500--12 .mu.M final) or NADH
(200--6 .mu.M final).
[0263] Construction of Site-Saturation Libraries (Generation
1):
[0264] One-site site-saturation libraries (with NNK codons) were
constructed using standard SOE PCR with Phusion polymerase, pGV3281
as template, and respective primer pairs #1-18. The fragments were
DpnI digested for 1 h, separated on an agarose gel,
freeze'n'squeeze (BIORAD) treated, and finally precipitated with
pellet paint (Novagen). The clean fragments served as templates for
the assembly PCRs using commercial T7 forward and reverse primers
(primers #1 and 2) as flanking primers. After successful assembly,
the insert was restriction digested with NdeI and XhoI, ligated
into pET22b(+), and electro-competent BL21(D3) cells (Lucigen) were
transformed.
[0265] Construction of Recombination Library (Generation 2):
[0266] The recombination library was constructed using SOE PCR
introducing mutations found at eight target sites while allowing
for the respective wild-type residues as well. We generated four
fragments using pGV3281 as template and primers #1, 2, and 19-32.
Primers 19 through 21, 22-24, 25-28, and 29-32 were mixed manually
to give equimolar distributions of the codons they contained. The
fragments were DpnI digested for 1 h at 37.degree. C., separated on
an agarose gel, freeze'n'squeezed (BIORAD) and finally pellet
painted (Novagen). The fragments served as templates in the
assembly PCR using commercial T7 forward and reverse primers as
flanking primers. The purified assembly product (Zymo clean up) was
restriction digested with NdeI and XhoI, ligated into pET22b(+),
and electro-competent BL21(D3) cells (Lucigen) were transformed.
Although the S53D mutation had not been identified as beneficial in
our NNK libraries, it was included in the recombination library
(=library 2). An assembly product of the recombination library
described above was used to introduce S53D via SOE PCR using primer
pairs 33-40 (Table 11). The forward and reverse primers were mixed
manually as described above. The resulting fragments were gel
purified and used as templates for the assembly PCR with flanking
T7 primers. The resulting assembly PCR product was treated as
described above.
[0267] Construction of Double NNK Library (K52NNKS53NNK)
(Generation 3):
[0268] The double NNK library was constructed via SOE PCR using
construct pETLI1G2 as template and primers #1 and 2, and 41 and 42.
The construction of the library was as described above
(site-saturation library). 2,800 colonies were picked for
screening.
[0269] High-Throughput Expression of L. lactis KARI Variants in E.
coli:
[0270] For growth and expression of KARI variants in deep well
plates, sterile toothpicks were used to pick single colonies into
shallow 96-well plates filled with 300 .mu.L LB.sub.amp. Fifty
.mu.L of these overnight cultures were used to inoculate deep well
plates filled with 600 .mu.L of LB.sub.amp per well. The plates
were grown at 37.degree. C. with shaking at 250 rpm for 3 h. One
hour before induction with IPTG (final concentration 0.5 mM), the
temperature of the incubator was reduced to 25.degree. C. After
induction, growth and expression continued for 20 h at 25.degree.
C. and 250 rpm. Cells were harvested at 5.300 g and 4.degree. C.
and then stored at -20.degree. C. The plates always contained four
wild-type or parent L. lactis KARI colonies, three BL21(DE3)
colonies carrying pET22b(+) to control for background reactions in
cell lysates, and one well that contained only media to make sure
the plates were free of contaminations.
[0271] High-Throughput Screening:
[0272] Frozen cell pellets were thawed at room temperature for 20
min and then 200 .mu.L of lysis buffer (100 mM Kpi, 750 mg/L
lysozyme, 10 mg/L DNaseI, pH 7) were added. Plates were vortexed to
resuspend the cell pellets. After a 60 min incubation phase at
37.degree. C. and shaking at 130 rpm, plates were centrifuged at
5,300 g and 4.degree. C. for 10 min. Forty .mu.L of the resulting
crude extracts were transferred into assay plates (flat bottom,
Rainin) using a liquid handling robot. Twenty mL assay buffer per
plate were prepared (100 mM Kpi, pH 7, 2.5 mM (S)-2-acetolactate, 1
mM DTT, 200 .mu.M NADPH or NADH, and 10 mM MgCl.sub.2) and 160
.mu.L thereof were added to each well to start the reaction
resulting in a 20% dilution of the ingredients. The depletion of
NAD(P)H was monitored at 340 nm in a plate reader (TECAN) over 200
s.
[0273] Results:
[0274] The residues chosen to test by site-saturation mutagenesis
were Y25, V48, R49, G51, S53, L85, and 189 of the L. lactis KARI
(SEQ ID NO: 2). Site-saturation libraries were constructed as
described in the materials and method section. After successful
transformation of BL21(DE3) cells, 88 individual clones per library
were chosen. The libraries were screened with NADH (not NADPH) as
cofactor. Screening results are summarized in Table 12.
TABLE-US-00016 TABLE 12 Exemplary Variants of Generation 1 NNK
Libraries. L. lactic Beneficial % improvement KARI Sites Mutations
over parent Y25 none n/a V48 P 73% V 44% L 80% R49 S 67% P 60% G51
none n/a K52 L 24% S53 none n/a L85 T 61% A 61% I89 A 43%
[0275] No improved variants were found in libraries harboring Y25,
G51, and S53 mutations.
[0276] Recombination Libraries:
[0277] Generation 2: A recombination library introducing all
mutations found at each site (Table 12) was constructed and also
allowing for the wild-type residues as well using pGV3281 as
template and primers #19-40. In addition, the S53D mutation was
tested in the context of the recombination library. However, given
previous experiences with this mutation (low expression levels, no
switch in cofactor specificity, and loss of activity) the
recombination library was constructed first, which only contained
the mutations found in Generation 1. This was deemed library 1.
Next, another round of SOE PCR was used to introduce S or D at
position 53 (library 2). Both libraries were separately ligated and
transformed. 1.100 of library 1 and 1,700 of library 2 were
screened for improved NADH consumption. Having no NADPH screening
data for the 2,800 clones and assuming that a switch in cofactor
specificity comes with the likely cost of losing activity on NADH,
selected variants with improved activity or activity equal to the
parent, as well variants that had lost up to 20% of their activity
on NADH were rescreened. In total, 88 variants were rescreened on
both cofactors narrowing the number down to 14 shown in Table 13.
Five double mutants 1A9, 1C2, 1G2, 2A9, and 1G5 were found. The
rest were single mutants. None of the 14 showed a switch in
cofactor specificity
TABLE-US-00017 TABLE 13 Exemplary Hits in Rescreen of Generation 2
Recombination Library. NADH/ Improve- NADPH ment in activity NADH
ratio activity Variant in screen [%] V48 R49 K52 S53 L85 I89 Parent
0.34 -- -- -- -- -- -- -- 1A9 0.52 71 -- L -- -- A -- 1C2 0.73 64 A
-- -- -- A -- 1E1 0.66 70 -- P -- -- -- -- 1E3 0.83 103 -- -- -- --
A -- 1G2 0.87 77 L P -- -- -- -- 1G5 0.7 70 -- -- -- -- A A 1G10
0.68 82 -- -- -- -- A -- 1H1 0.9 86 -- -- -- -- A -- 2A5 0.73 72.5
-- V -- -- -- -- 2A8 0.51 55 -- P -- -- -- -- 2A9 0.5 52.5 L P --
-- -- -- 2A10 0.52 59 -- V -- -- -- -- 2E5 0.71 34.5 L -- -- -- --
-- 2G8 0.72 80 -- V -- -- -- --
[0278] Variants 1A9, 1C2, 1G2, and 1G5 were expressed, purified,
and characterized (Table 14). Mutation L85A is noteworthy. In all
three cases (1A9, 1C2, and 1G5), the NADPH K.sub.M value was either
cut in half (1A9) or below 1 .mu.M (1C2 and 1G5) and thus beneath
the measurable threshold. These variants also showed the lowest
NADH K.sub.M values with 99, 115, and 112 .mu.M. Mutation L85A
could be conceived as being generally activating.
TABLE-US-00018 TABLE 14 Comparison of L. lactis KARI and Variants
Thereof mutations U/mg K.sub.m [M] for cofactor Gen Variant (gene)
V48 R49 K52 S53 L85 I89 NADH NADPH ratio NADH NADPH 0
LI_KARI-.sup.his6 (wt) V R K S L I 0.05 0.34 .+-. 0.05 0.15 285
.+-. 30 13 .+-. 1.3 2 LI_KARI.sup.1A9-his6 L A 0.2 0.33 0.6 99 8
LI_KARI.sup.1G2-his6 A A 0.17 0.33 0.5 115 <1
LI_KARI.sup.1G2-his6 L P 0.21 0.34 0.6 150 13 LI_KARI.sup.1G5-his6
A A 0.21 0.4 0.5 112 <1 Gen Variant (gene) k.sub.cat.sup.[s-1]
k.sub.cat/K.sub.m [M.sup.-1*s.sup.-1] 0 LI_KARI-.sup.his6 (wt) NADH
NADPH NADH NADPH ratio 2 LI_KARI.sup.1A9-his6 0.1 0.8 530 65,000
0.008 LI_KARI.sup.1G2-his6 0.5 0.8 5,000 102,000 0.049
LI_KARI.sup.1G2-his6 0.4 0.8 3,700 >800,000 <0.005
LI_KARI.sup.1G5-his6 0.5 0.8 3,300 65,000 0.051 0.5 1.0 4,700
>990,000 <0.005
[0279] None of the variants had mutations at residues K52 and S53,
positions that bind the NADPH phosphate with high probability.
Based on the results of Generation 1, it was hypothesized that the
strong NADPH binding is not due to one residue only, but rather due
to a concerted binding via R49, K52, and S53. Disrupting the
binding of R49 with a mutation such as R49P would give the
opportunity to explore different combinations at the two important
sites 52 and 53. Thus, a double NNK library at these two positions
was generated using a variant with a mutation at residue R49 as
template.
[0280] Generation 3: Double NNK Library at Positions K52 and
S53:
[0281] Given the data presented in Table 14, the choice of the
parent for the next round was between 1A9 and 1G2. Both had a
mutation at position R49 (L and P); in addition, 1A9 carried the
L85A mutation described above; 1G2 had a mutation at position 48
(V48L). Even though the NADH K.sub.M value and catalytic efficiency
of 1G2 were not as favorable as 1A9's, 1G2 was chosen as parent
because, due to the lack of L85A, its NADPH activities were
parent-like. Thus, its improvements stem from increased NADH
activity only.
[0282] A library with 1G2 as parent was generated using primers #41
and 42 and the commercial T7 primers (#1 and 2). Approximately
2,800 individual colonies were screened for both NADH and NADPH
consumption. The introduction of two negative charges, K52E and
S53D, gives the highest NADH/NADPH ratio in the screen (variant
4H8). However, when the order of the residues is reversed, K52D and
S53E (variant 3H5), the protein has potential folding issues. The
introduction of one negative charge in combination with L, P, S, or
K results in at least four-fold ratio improvements compared to
parent 1G2. However, when K52 is mutated to P and S53 is still able
to bind phosphate, no beneficial effects on the ratio were observed
(variant 2G6).
TABLE-US-00019 TABLE 15 NADH/NADPH activity ratio measured in
screen of double NNK library. The variants in this table are the
top hits (except 2G6) of 1/6.sup.th of this library. NADH/ NADPH
Variant in screen K52 S53 LI_KARI.sup.1G-his6 0.5 K S 4H8 >10 E
D 3C7 8.2 L D 3H5 8.2 D E 3E9 6.1 P E 2A4 3.7 K E 3F9 2.2 S D 2G6
0.5 P S
[0283] Four out of eight variants are shown in Table 15 (4H8, 3C7,
3H5, and 3E9). In Table 16, characterization data of the cofactor
switched variants is presented. Each is compared to the wild-type
L. lactis KARI and variants found in Generation 2. The NADPH
K.sub.M value of LI_KARI.sup.3C7-his6 is estimated to be greater
than 1000 .mu.M. An NADPH concentration of 500 .mu.M in the cuvette
in the spectrometer was capable of being measured. At this
concentration, the variant had not reached saturation yet. The
other two variants, 3H5 and 3E9, had double peaks in the
purification chromatogram, low expression levels, and also very low
activity after purification.
TABLE-US-00020 TABLE 16 Comparison of properties of
LI_KARI.sup.his6 and Generation 2 and Generation 3 enzyme variants.
mutations U/mg K.sub.m [.mu.M] for cofactor Gen Variant (gene) V48
R49 K52 S53 L85 I89 NADH NADPH ratio NADH NADPH 0 LI_KARI-.sup.his6
(wt) V R K S L I 0.05 0.34 .+-. 0.05 0.15 285 .+-. 30 13 .+-. 1.3 2
LI_KARI.sup.1A9-his6 L A 0.2 0.33 0.6 99 8 LI_KARI.sup.1C2-his6 A A
0.17 0.33 0.5 115 <1 LI_KARI.sup.1G2-his6 L P 0.21 0.34 0.6 150
13 LI_KARI.sup.1G5-his6 A A 0.21 0.4 0.5 112 <1 3
LI_KARI.sup.4H8-his6 L P E D 0.14 0.024 5.8 128 .+-. 9 1180 .+-.
280 LI_KARI.sup.3C1-his6 L P L D 0.16 0.003 53.3 108 .+-. 9
>1000 k.sub.cat [.sup.s-1] k.sub.cat/K.sub.m [M.sup.-1*s.sup.-1]
Gen Variant (gene) NADH NADPH NADH NADPH ratio 0 LI_KARI-.sup.his6
(wt) 0.1 0.8 530 65,000 0.008 2 LI_KARI.sup.1A9-his6 0.5 0.8 5,000
102,000 0.049 LI_KARI.sup.1C2-his6 0.4 0.8 3,700 >800.000
<0.005 LI_KARI.sup.1G2-his6 0.5 0.8 3,300 65,000 0.051 3
LI_KARI.sup.1G5-his6 0.5 1.0 4,700 >990,000 <0.005
LI_KARI.sup.4H8-his6 0.35 0.06 2,700 <50 54 LI_KARI.sup.3C1-his6
0.4 0.01 3,700 <7 529
[0284] Generation 3 variants LI_KARI.sup.4H8-his6 and
LI_KARI.sup.3C7-his6 exhibit switches in cofactor specificities for
NADH over NADPH in terms of catalytic efficiency. Both variants
carry four mutations and only differ at position K52 (K52E or K52L,
respectively). Residues K52 and S53D appear to be important
determinants of cofactor specificity. Both variants have .about.2.5
fold reduced NADH K.sub.M values relative to the wild-type L.
lactis KARI. Both variants have lost almost all activity (U/mg) on
NADPH: 14-fold decrease of activity for 4H8 and 113-fold decrease
of activity for 3C7.
[0285] In addition to the above-described modifications, further
generations of mutants were constructed. Briefly, a recombination
library was constructed using standard overlap extension polymerase
chain reaction and primer pairs 1, 2, and 43-54. In these
additional generations, two variants exhibited beneficial
properties: LI_NKR.sup.Gen6a-his6 and LI_NKR.sup.Gen6b-his6.
LI_NKR.sup.Gen6a-his contained mutations K118E, T182S, and E320K
and showed a 3.7-fold increase in catalytic efficiency in the
presence of NADH and a 15-fold decrease in 2S-AL K.sub.M value.
LI_NKR.sup.Gen6b-his retained mutations E59K, T182S, and E320K and
had an almost 40-fold improved K.sub.M value for 2S-AL. The
catalytic efficiency of this enzyme in the presence of NADH was
14.8-fold increased compared to its parent, LI_NKR.sup.Gen3-his6.
LI_NKR.sup.Gen6b-his showed a complete switch of cofactor
preference. The characterization data is summarized in Table
17.
TABLE-US-00021 TABLE 17 Characterization of recombination variants
LI_NKR.sup.Gen6a-his and LI_NKR.sup.Gen6b-his in comparison to
their lineage. K.sub.m [.mu.M] for Mutations U/mg cofactor Gen
Variant (gene) V48 R49 K52 S53 E59 K118 T182 E320 NADH NADPH ratio
NADH NADPH 0 LI_KARI.sup.his6 (wt) 0.5 0.34 .+-. 0.05 0.15 285 .+-.
30 13 .+-. 1.3 3 LI_NKR.sup.3C7-his6 L P L D 0.16 .+-. 0.03 .+-. 5
.+-. 108 .+-. 1000 .+-. 0.002 0.004 0.7 9 100 6
LI_NKR.sup.Gen6a-his6 L P L D E S K 0.25 .+-. 0.16 .+-. 1.56 .+-.
45 .+-. 1059 .+-. 0.003 0.007 0.1 19 306 6 LI_NKR.sup.Gen6b-his6 L
P L D K S K 0.43 .+-. 0.14 .+-. 3 .+-. 15 .+-. 749 .+-. 0.01 0.031
0.7 4 95 K.sub.m K.sub.i [mM] [mM] for for R- IC.sub.50 Gen Variant
(gene) k.sub.cat[s.sup.-1] k.sub.cat/K.sub.m[ M.sup.-1*s.sup.-1]
substrate DHIV K.sub.i/K.sub.M (mM) for 0 LI_KARI.sup.his6 (wt)
NADH NADPH NADH NADPH ratio NADH NADH NADH R-DHIV 3
LI_NKR.sup.3C7-his6 0.1 0.8 .+-. 430 .+-. 65,000 .+-. 0.007 .+-.
5.6 .+-. 1.7 0.3 N.D 0.12 45 11559 0.0 1.6 6 LI_NKR.sup.Gen6a-his6
0.4 .+-. 0.08 .+-. 3,704 .+-. 80 .+-. 46 .+-. 8.2 .+-. N.D N.D N.D
0.005 0.01 312 13 9 1.0 6 LI_NKR.sup.Gen6b-his6 0.62 .+-. 0.4 .+-.
13,808 .+-. 375 .+-. 37 .+-. 0.53 .+-. 0.05 .+-. 0.1 1.05 .+-. 0.01
0.02 5,834 110 19 0.11 0.011 0.26 1.07 .+-. 0.35 .+-. 71,248 .+-.
465 .+-. 153 .+-. 0.21 .+-. 0.01 .+-. 0.05 0.26 .+-. 0.03 0.08
19,102 122 57 0.03 0.001 0.06
Example 4
NADH-Dependent KARI Derived from Shewanella and Salmonella
[0286] The following example illustrates exemplary long-form KARI
enzymes from Shewanella sp. and Salmonella enterica and
corresponding NADH-dependent ketol-acid reductoisomerases (NKR)
derived therefrom.
[0287] Plasmids and primers disclosed in this example are shown in
Tables 18-19 below.
TABLE-US-00022 TABLE 18 Plasmids Disclosed in Example 4. Plasmids
Genotype pET22b(+) PT7, bla, ori pBR322, lacI, C-term 6xHis pGV3195
PT7::Se1_KARI.sup.his6, bla, oripBR322, lacI pGV3627
PT7::Sh_sp_KARI_coSc.sup.his6, bla, oripBR322, lacI pGV3628
PT7::Sh_sp_NKR_coSc.sup.DDhis6, bla, oripBR322, lacI pGVSh_sp_S78D
PT7::Sh_sp_KARI_coSc.sup.S78Dhis6, bla, oripBR322, lacI pGV3629
PT7::Sh_sp_NKR_coSc.sup.6E6his6, bla, oripBR322, lacI pGV3630
PT7::Se2_KARI_coSc.sup.his6, bla, oripBR322, lacI pGVSe2_S78D
PT7::Se2_KARI_coSc.sup.S78Dhis6, bla, oripBR322, lacI pGV3631
PT7::Se2_NKR_coSc.sup.DDhis6, bla, oripBR322, lacI pGV3632
PT7::Se2_NKR_coSc.sup.6E6his6, bla, oripBR322, lacI
TABLE-US-00023 TABLE 19 Oligonucleotide Primers Disclosed in
Example 4. Primer name Sequence Sh_S78D_for
GCACAAAAGAGAGCCGATTGGCAAAAAGCGAC (SEQ ID NO: 145) Sh_S78D_rev
GTCGCTTTTTGCCAATCGGCTCTCTTTTGTGC (SEQ ID NO: 146) Se2_S78D_for
GCAGAAAAGAGAGCCGATTGGCGTAAAGCGACGGA (SEQ ID NO: 147) Se2_S78D_rev
TCCGTCGCTTTACGCCAATCGGCTCTCTTTTCTGC (SEQ ID NO: 148)
[0288] Mutations relative to wild-type Salmonella enterica KARI
(Se2_KARI) and Shewanella sp. KARI (Sh_sp_KARI) are listed in Table
20 below.
TABLE-US-00024 TABLE 20 Mutations Relative to Se2_KARI and Sh_KARI.
Source Variant (enzyme) Mutations Salmenella Se2_KARI n/a enterica
Se2_KARI.sup.S78D S78D Se2_NKR.sup.DD R76D, S78D Se2_NKR.sup.6E6
A71S, R76D, S78D, Q110V Shewanella sp. Sh_sp_KARI n/a
Sh_sp_NKR.sup.S78D S78D Sh_sp_NKR.sup.DD R76D, S78D
Sh_sp_NKR.sup.6E6 A71S, R76D, S78D, Q110V
[0289] Genes encoding Se2_KARI, Se2_NKR.sup.DD, Se2_NKR.sup.6E6,
Sh_sp_KARI, Sh_sp_NKR.sup.DD, and Sh_sp_NKR.sup.6E6 were
synthesized by GenScript USA Inc. (Piscataway, N.J. 08854 USA) with
flanking NdeI and XhoI sites. The genes were isolated by
restriction enzyme digestion with NdeI and XhoI for 1 hour at
37.degree. C. The expression vector, pGV3195, was also digested
with NdeI and XhoI for 1 hour at 37.degree. C. The fragments were
ligated using T4 DNA ligase from New England Biolabs (Ipswich,
Mass. USA). The ligated DNAs were transformed into chemically
competent E. coli DH5.alpha. cells, incubated for 1 h at 37.degree.
C. in SOC medium, and plated to LB.sub.amp agar plates (Luria
Bertani Broth, Research Products International Corp, supplemented
with 100 .mu.g/mL ampicillin) to yield single colonies. After
confirming the correct sequence, E. coli BL21(DE3) cells were
transformed with the correct plasmids for expression.
[0290] Genes encoding Se2_KARI.sup.S78D and Sh_sp_NKR.sup.S78D:
Single aspartic acid substitutions were introduced using the
QuikChange site-directed mutagenesis kit according to
manufacturer's protocol (Stratagene). Plasmids pGV3627 and pGV3630
encoding Sh_sp_KARI and Se2_KARI, respectively, were used as
templates. The respective primer pairs were primers Sh_S78D_for and
Sh_S78D_rev and primers Se2_S78D_for and Se2_S78D_rev. Pfu turbo
polymerase (Stratagene) was used as the polymerase in the following
PCR program: 95.degree. C. for 2 min; 95.degree. C. for 30 s,
55.degree. C. for 30 s, 72.degree. C. for 8 min (repeat 15 times);
72.degree. C. for 10 min. After the PCR program was completed, the
reaction mixtures were digested with DpnI for 1 h at 37.degree. C.
Then, chemically competent E. coli XL1-Gold cells were transformed
with 3 .mu.L of the un-cleaned PCR mixtures and the cells were
allowed to recover in SOC medium at 37.degree. C. with shaking at
250 rpm for 1 h. The recovery allowed the cells to close the
nick-containing DNA produced during the PCR and thus to generate
circularized plasmids. We then plated varying volumes on LB.sub.amp
agar plates (Luria Bertani Broth, Research Products International
Corp, supplemented with 100 .mu.g/mL ampicillin) to yield single
colonies. After confirming the correct sequence, E. coli BL21(DE3)
cells were transformed with the correct plasmids for
expression.
[0291] Heterologous expression of Se2_KARI and Sh_sp_KARI variants
in E. coli: The expression of Se2_KARI, Sh_sp_KARI, and their
corresponding NKR variants (Table 20) was conducted in 0.5 L
Erlenmeyer flasks filled with 0.2 L LB.sub.amp (Luria Bertani
Broth, Research Products International Corp, supplemented with 100
.mu.g/mL ampicillin) inoculated with overnight culture to an
initial OD.sub.600 of 0.1. After growing the expression cultures at
37.degree. C. with shaking at 250 rpm for 4 h, the cultivation
temperature was dropped to 25.degree. C., and KARI expression was
induced with IPTG to a final concentration of 0.5 mM. After 24 h at
25.degree. C. and shaking at 250 rpm, the cells were pelleted at
5,300 g for 10 min and then frozen at -20.degree. C. until further
use.
[0292] Sh_sp_KARI and its NKR variants were expressed in 200-mL
cultures and purified over a 1-mL histrap HP column. The K.sub.M,
k.sub.cat, and specific activity values were measured as described
above, and the results are summarized in Table 21. In terms of the
ratio of catalytic efficiency with NADH over NADPH, variants
Sh_sp_NKR.sup.S78D, Sh_sp_NKR.sup.R76DS78D, and Sh_sp_NKR.sup.6E6
can be defined as being NADH-dependent KARIs (NKR) in terms of
their catalytic efficiencies.
[0293] Se2_KARI and its NKR variants were expressed in 200-mL
cultures and purified over a 1-mL histrap HP column. The K.sub.M,
k.sub.cat, and specific activity values were measured as described
above, and the results are summarized in Table 22. In terms of the
ratio of catalytic efficiency with NADH over NADPH, variants
Se2_NKR.sup.DD and Se2_NKR.sup.6E6 can be defined as being
NADH-dependent KARIs (NKR). Additional mutations at positions D146,
G185, and K433 are generally expected to further improve activity
of the Se2_KARI (data not shown).
TABLE-US-00025 TABLE 21 Comparison of properties of wild-type
Shewanella sp. KARI (Sh_sp_KARI), the single and double aspartic
acid variants, and the "6E6" variant. Data is based on measurements
using purified proteins. Sp. Activity [U/mg] K.sub.m [.mu.M] for
cofactor k.sub.cat[.sup.s-1] NADH .+-. NADPH .+-. ratio .+-. NADH
.+-. NADPH .+-. NADH .+-. NADPH .+-. Sh_sp_KARI 0.30 0.012 1.2
0.013 0.2 0.0 415 44 .ltoreq.1 1.1 0.05 4.5 0.05 Sh_sp_NKR.sup.S78D
0.36 0.024 0.5 0.042 0.7 0.1 130 15 267 26 1.3 0.09 2.0 0.16
Sh_sp_NKR.sup.DD 0.34 0.001 0.03 0.004 10.7 1.5 90 24 >1000 1.3
0.01 0.1 0.02 Sh_sp_NKR.sup.6E6 0.66 0.00 0.1 0.013 9.1 1.7 75 10
600 130 2.4 0.00 0.3 0.05 k.sub.cat/K.sub.m[M.sup.-1*s.sup.-1] NADH
.+-. NADPH .+-. ratio .+-. Sh_sp_KARI 2,649 301 4,479,890 0.0006
Sh_sp_NKR.sup.S78D 10,269 1,360 7,373 924 1.4 0.3 Sh_sp_NKR.sup.DD
14,022 3,740 <117 119 Sh_sp_NKR.sup.6E6 32,410 4,322 446 127 73
22.9
TABLE-US-00026 TABLE 22 Comparison of properties of wild-type
Salmonella enterica KARI (Se2_KARI), the single and double aspartic
acid variants, and the "6E6" variant. Data is based on measurements
using purified proteins. Sp. Activity [U/mg] K.sub.m [.mu.M] for
cofactor k.sub.cat[.sup.s-1] NADH .+-. NADPH .+-. ratio .+-. NADH
.+-. NADPH .+-. NADH .+-. NADPH .+-. Se2_KARI 0.14 0.009 1.1 0.062
0.1 0.0 157 4 8 2 0.51 0.03 3.96 0.23 Se2_NKR.sup.S78D 0.17 0.005
0.4 0.015 0.4 0.0 233 43 272 23 0.63 0.02 1.47 0.05 Se2_NKR.sup.DD
0.37 0.002 0.03 0.005 12.4 1.9 121 20 >1000 1.36 0.01 0.11 0.02
Se2_NKR.sup.6E6 0.64 0.10 0.1 0.045 6.6 3.2 24 4 630 291 2.33 0.35
0.36 0.01 k.sub.cat/K.sub.m[M.sup.-1*S.sup.-1] NADH .+-. NADPH .+-.
ratio .+-. Se2_KARI 3,229 222 495,409 127,118 0.01 0.0
Se2_NKR.sup.S78D 2,696 503 5,402 498 0.5 0.1 Se2_NKR.sup.DD 11,222
1,856 <110 >102 Se2_NKR.sup.6E6 97,284 21.857 565 261 172
88.5
[0294] The foregoing detailed description has been given for
clearness of understanding only and no unnecessary limitations
should be understood there from as modifications will be obvious to
those skilled in the art.
[0295] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.
[0296] The disclosures, including the claims, figures and/or
drawings, of each and every patent, patent application, and
publication cited herein are hereby incorporated herein by
reference in their entireties.
Sequence CWU 1
1
14811479DNAShewanella sp. 1atggctaact attttaactc tctgaattta
cgtcaacaat tagaacagct tggccaatgc 60cgttttatgg atcgctccga gtttagcgat
ggctgcaatt acatcaaaga ttggaatatt 120gttattttag gttgtggtgc
tcagggtctt aaccaaggtc tgaacatgcg tgactctggg 180ctgaatattg
cctacgccct gcgcccagaa gccattgccc agaaacgtgc ctcatggcaa
240aaagccacgg acaacggctt taaagtgggc acctttgaag agctgatccc
aacggcggat 300ttggtactga acttaacgcc cgataagcag cactccaatg
tggttagcgc tgtgatgcca 360ctgatgaaac aaggcgcaac gctgtcttat
tcccacggtt ttaacatcgt tgaagaaggc 420atgcagatcc gccccgatat
tacagttgtg atggtagcgc ctaagtgccc aggtactgaa 480gtgcgtgaag
aatacaagcg tggttttggt gtaccgacac tgattgcagt gcacccagaa
540aacgacccta atggcgatgg tttagagatt gccaaagcct atgcgagtgc
cacaggcggt 600gaccgcgctg gcgtgttgca atcttccttt attgccgaag
taaaatcgga tctgatgggc 660gaacaaacca ttctgtgcgg tatgttgcag
acgggcgcta tcctaggtta cgacaagatg 720gtggccgatg gtgttgaacc
tggctatgcc gctaagttaa tccaacaagg ttgggaaacc 780gtgaccgagg
cgcttaagca cggcggtatc accaacatga tggacagact gtctaatcct
840gccaagatta aagcgtttga aattgcagaa gatttaaaag aaattctgca
acccttattc 900gaaaaacata tggatgacat catcagcggc gagttctcac
gcactatgat gcaagactgg 960gcgaatgacg acgctaacct gctgcgttgg
cgcgccgaaa ccgccgaaac gggcttcgaa 1020aatgcccctg tttcgagcga
gcatatcgac gagcaaacct atttcgacaa ggggattttc 1080ctagttgcga
tgatcaaagc cggtgtcgaa ttagccttcg atactatggt gtcggcgggt
1140attgttgaag agtcagccta ttacgaatca ctgcatgaaa cgccgctgat
cgctaacacc 1200atcgcccgta aacgtcttta cgagatgaac gtggtgatct
cggataccgc agaatacggt 1260tgttatctgt tcaaccatgc tgcagtacct
atgctgcgtg actatgtaaa tgccatgtcg 1320ccagagtatt taggtgcggg
tctgaaggac agttctaaca acgtcgataa cctgcagtta 1380atcgccatca
atgatgcgat tcgccacact tcagttgagt atatcggtgc ggaacttcgt
1440ggttatatga ctgatatgaa aagtattgtt ggagcttaa
14792492PRTShewanella sp. 2Met Ala Asn Tyr Phe Asn Ser Leu Asn Leu
Arg Gln Gln Leu Glu Gln 1 5 10 15 Leu Gly Gln Cys Arg Phe Met Asp
Arg Ser Glu Phe Ser Asp Gly Cys 20 25 30 Asn Tyr Ile Lys Asp Trp
Asn Ile Val Ile Leu Gly Cys Gly Ala Gln 35 40 45 Gly Leu Asn Gln
Gly Leu Asn Met Arg Asp Ser Gly Leu Asn Ile Ala 50 55 60 Tyr Ala
Leu Arg Pro Glu Ala Ile Ala Gln Lys Arg Ala Ser Trp Gln 65 70 75 80
Lys Ala Thr Asp Asn Gly Phe Lys Val Gly Thr Phe Glu Glu Leu Ile 85
90 95 Pro Thr Ala Asp Leu Val Leu Asn Leu Thr Pro Asp Lys Gln His
Ser 100 105 110 Asn Val Val Ser Ala Val Met Pro Leu Met Lys Gln Gly
Ala Thr Leu 115 120 125 Ser Tyr Ser His Gly Phe Asn Ile Val Glu Glu
Gly Met Gln Ile Arg 130 135 140 Pro Asp Ile Thr Val Val Met Val Ala
Pro Lys Cys Pro Gly Thr Glu 145 150 155 160 Val Arg Glu Glu Tyr Lys
Arg Gly Phe Gly Val Pro Thr Leu Ile Ala 165 170 175 Val His Pro Glu
Asn Asp Pro Asn Gly Asp Gly Leu Glu Ile Ala Lys 180 185 190 Ala Tyr
Ala Ser Ala Thr Gly Gly Asp Arg Ala Gly Val Leu Gln Ser 195 200 205
Ser Phe Ile Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile 210
215 220 Leu Cys Gly Met Leu Gln Thr Gly Ala Ile Leu Gly Tyr Asp Lys
Met 225 230 235 240 Val Ala Asp Gly Val Glu Pro Gly Tyr Ala Ala Lys
Leu Ile Gln Gln 245 250 255 Gly Trp Glu Thr Val Thr Glu Ala Leu Lys
His Gly Gly Ile Thr Asn 260 265 270 Met Met Asp Arg Leu Ser Asn Pro
Ala Lys Ile Lys Ala Phe Glu Ile 275 280 285 Ala Glu Asp Leu Lys Glu
Ile Leu Gln Pro Leu Phe Glu Lys His Met 290 295 300 Asp Asp Ile Ile
Ser Gly Glu Phe Ser Arg Thr Met Met Gln Asp Trp 305 310 315 320 Ala
Asn Asp Asp Ala Asn Leu Leu Arg Trp Arg Ala Glu Thr Ala Glu 325 330
335 Thr Gly Phe Glu Asn Ala Pro Val Ser Ser Glu His Ile Asp Glu Gln
340 345 350 Thr Tyr Phe Asp Lys Gly Ile Phe Leu Val Ala Met Ile Lys
Ala Gly 355 360 365 Val Glu Leu Ala Phe Asp Thr Met Val Ser Ala Gly
Ile Val Glu Glu 370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His Glu Thr
Pro Leu Ile Ala Asn Thr 385 390 395 400 Ile Ala Arg Lys Arg Leu Tyr
Glu Met Asn Val Val Ile Ser Asp Thr 405 410 415 Ala Glu Tyr Gly Cys
Tyr Leu Phe Asn His Ala Ala Val Pro Met Leu 420 425 430 Arg Asp Tyr
Val Asn Ala Met Ser Pro Glu Tyr Leu Gly Ala Gly Leu 435 440 445 Lys
Asp Ser Ser Asn Asn Val Asp Asn Leu Gln Leu Ile Ala Ile Asn 450 455
460 Asp Ala Ile Arg His Thr Ser Val Glu Tyr Ile Gly Ala Glu Leu Arg
465 470 475 480 Gly Tyr Met Thr Asp Met Lys Ser Ile Val Gly Ala 485
490 31485DNAVibrio fischeri 3atgtctaact actttaatac gctaaattta
cgtgaacaat tagatcaact aggtcgttgt 60cgctttatgg atcgtgaaga atttgcaaca
gaagctgatt accttaaagg taaaaaagtg 120gttattgttg gttgtggtgc
tcaaggctta aaccaaggcc ttaatatgcg tgattcaggc 180ttagatgttg
cttatgcact gcgtcaagcc gctattgatg agcaacgaca atcttataaa
240aatgcaaaag aaaatggttt tgaagtagct agctatgaaa ctctgatccc
tcaagctgac 300ctagttatta atcttactcc tgataaacaa catactaatg
tagttgaaac tgttatgcct 360ctaatgaaag agggggcagc tttaggctat
tcacatggtt ttaatgttgt tgaagaaggg 420atgcaaatcc gtaaagattt
gacggttgtt atggttgctc ctaagtgtcc aggaacagaa 480gttcgtgaag
aatataaacg tggttttggg gttccaactc taattgctgt tcacccagaa
540aatgatccta aaggtgaagg ttgggatatt gctaaggctt gggctgctgg
tacaggtggt 600caccgtgcgg gttgtctaga gtcttctttt gtcgctgaag
ttaaatctga ccttatgggt 660gagcaaacga ttctttgtgg catgctacaa
gctggttcta ttgtatctta cgagaagatg 720attgctgacg gtattgagcc
tggttatgca ggtaaacttc tacagtacgg ttgggaaaca 780attactgaag
ccctaaagtt tggcggtgtt acgcatatga tggatcgcct ttcaaaccca
840gctaaagtaa aagcttttga gctttcagaa gaacttaaag agctaatgcg
tccactttat 900aataagcata tggacgatat tatttctggt gagttttctc
gcacaatgat ggctgattgg 960gctaatgatg atgttaatct atttggctgg
cgtgaagaaa caggtcaaac tgcgtttgaa 1020aactaccctg agtctgatgt
agagatctct gaacaagaat actttgataa cggtatttta 1080ctcgttgcaa
tggttcgtgc cggcgttgaa ttagcttttg aagctatgac tgcatcaggc
1140attatcgatg aatcagctta ctatgagtcg ctgcacgaat taccactgat
tgctaacact 1200gtagcacgta agcgtttgta cgaaatgaac gtagttattt
ctgatactgc tgaatatggt 1260aactacctat ttgcaaatgt agcaacgcca
cttcttcgtg agaaattcat gccttctgtt 1320gaaacagatg ttattggccg
aggattaggt gaggcatcaa atcaagtgga taatgcaacg 1380ctaatcgctg
ttaatgatgc gattcgtaat catccagttg aatatattgg tgaagaatta
1440cgtagctaca tgagcgatat gaagcgaatt gcagttggtg gctaa
14854494PRTVibrio fischeri 4Met Ser Asn Tyr Phe Asn Thr Leu Asn Leu
Arg Glu Gln Leu Asp Gln 1 5 10 15 Leu Gly Arg Cys Arg Phe Met Asp
Arg Glu Glu Phe Ala Thr Glu Ala 20 25 30 Asp Tyr Leu Lys Gly Lys
Lys Val Val Ile Val Gly Cys Gly Ala Gln 35 40 45 Gly Leu Asn Gln
Gly Leu Asn Met Arg Asp Ser Gly Leu Asp Val Ala 50 55 60 Tyr Ala
Leu Arg Gln Ala Ala Ile Asp Glu Gln Arg Gln Ser Tyr Lys 65 70 75 80
Asn Ala Lys Glu Asn Gly Phe Glu Val Ala Ser Tyr Glu Thr Leu Ile 85
90 95 Pro Gln Ala Asp Leu Val Ile Asn Leu Thr Pro Asp Lys Gln His
Thr 100 105 110 Asn Val Val Glu Thr Val Met Pro Leu Met Lys Glu Gly
Ala Ala Leu 115 120 125 Gly Tyr Ser His Gly Phe Asn Val Val Glu Glu
Gly Met Gln Ile Arg 130 135 140 Lys Asp Leu Thr Val Val Met Val Ala
Pro Lys Cys Pro Gly Thr Glu 145 150 155 160 Val Arg Glu Glu Tyr Lys
Arg Gly Phe Gly Val Pro Thr Leu Ile Ala 165 170 175 Val His Pro Glu
Asn Asp Pro Lys Gly Glu Gly Trp Asp Ile Ala Lys 180 185 190 Ala Trp
Ala Ala Gly Thr Gly Gly His Arg Ala Gly Cys Leu Glu Ser 195 200 205
Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile 210
215 220 Leu Cys Gly Met Leu Gln Ala Gly Ser Ile Val Ser Tyr Glu Lys
Met 225 230 235 240 Ile Ala Asp Gly Ile Glu Pro Gly Tyr Ala Gly Lys
Leu Leu Gln Tyr 245 250 255 Gly Trp Glu Thr Ile Thr Glu Ala Leu Lys
Phe Gly Gly Val Thr His 260 265 270 Met Met Asp Arg Leu Ser Asn Pro
Ala Lys Val Lys Ala Phe Glu Leu 275 280 285 Ser Glu Glu Leu Lys Glu
Leu Met Arg Pro Leu Tyr Asn Lys His Met 290 295 300 Asp Asp Ile Ile
Ser Gly Glu Phe Ser Arg Thr Met Met Ala Asp Trp 305 310 315 320 Ala
Asn Asp Asp Val Asn Leu Phe Gly Trp Arg Glu Glu Thr Gly Gln 325 330
335 Thr Ala Phe Glu Asn Tyr Pro Glu Ser Asp Val Glu Ile Ser Glu Gln
340 345 350 Glu Tyr Phe Asp Asn Gly Ile Leu Leu Val Ala Met Val Arg
Ala Gly 355 360 365 Val Glu Leu Ala Phe Glu Ala Met Thr Ala Ser Gly
Ile Ile Asp Glu 370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His Glu Leu
Pro Leu Ile Ala Asn Thr 385 390 395 400 Val Ala Arg Lys Arg Leu Tyr
Glu Met Asn Val Val Ile Ser Asp Thr 405 410 415 Ala Glu Tyr Gly Asn
Tyr Leu Phe Ala Asn Val Ala Thr Pro Leu Leu 420 425 430 Arg Glu Lys
Phe Met Pro Ser Val Glu Thr Asp Val Ile Gly Arg Gly 435 440 445 Leu
Gly Glu Ala Ser Asn Gln Val Asp Asn Ala Thr Leu Ile Ala Val 450 455
460 Asn Asp Ala Ile Arg Asn His Pro Val Glu Tyr Ile Gly Glu Glu Leu
465 470 475 480 Arg Ser Tyr Met Ser Asp Met Lys Arg Ile Ala Val Gly
Gly 485 490 51476DNAGramella forsetii 5atgaccaact attttaacag
cctttcttta cgtgatcaat tagctcagct tggaacctgc 60aggtttatgg agctggatga
attcagcaac gaggtggctg tcctaaaaga taaaaaaatt 120gtgatcgtag
gctgtggagc ccagggtctt aatcaggggc tcaatatgcg cgatagcgga
180ctcgatatct catatgcgtt aagggaagga gcgattaaag aaaagcgaca
gtcctggaaa 240aatgctacgg aaaataattt taacgtagga acttatgagg
agcttattcc aaaggctgat 300cttgttatca atcttacgcc agataaacaa
catacttcgg tgatcaaggc gattcaacct 360catatcaaaa aagatgcggt
actttcttac tctcatggtt tcaacattgt ggaagaagga 420acgaagatac
gtgaagatat aacggtaatt atggtcgcgc caaaatgtcc cggaactgag
480gtgagggaag aatataaaag aggttttgga gtgccgactc ttatcgcggt
tcatccggaa 540aatgatcctc atggaattgg cctggattgg gcaaaagctt
atgcgtatgc tacaggtggt 600cacagggccg gagtactgga atcttctttt
gttgctgaag taaaatctga cctaatgggg 660gaacaaacaa tgctttgtgg
agttcttcaa acaggatcga tcttaacttt cgataaaatg 720gttgcagatg
gtgtggagcc aaattatgct gcaaaactta tccagtatgg atgggaaaca
780attactgaag ccctgaaaca tggtggaata accaatatga tggacaggct
ttcaaatcct 840gcaaagctta gagcgaatga aattgctgaa gaacttaaag
agaaaatgcg tccgcttttt 900cagaaacata tggatgatat aatttcagga
gaattcagca gtcgaatgat gcgtgactgg 960gcaaatgatg ataaagaatt
actcacctgg cgtgccgaaa cagagaatac cgcttttgaa 1020aaaactgaag
ccacttcaga agagatcaaa gagcaggaat attttgataa aggtgtgctg
1080atggtggcct ttgtaagggc aggtgtagag ctggcctttg aaacgatggt
ggaagccggg 1140ataattgaag aatcggctta ttatgaatca cttcatgaaa
ctccgcttat agccaatacc 1200attgccagaa agaaattata cgagatgaat
cgtgtgattt cagatactgc tgaatacggt 1260tgttatttat ttgatcatgc
tgcaaaacca ttggtgaaag attatgtaaa ctcacttgaa 1320ccggaagttg
ccgggaagaa atttggaaca gattgtaatg gtgtggataa ccagaaattg
1380atacacgtga atgatgatct tagaagtcat ccggttgaaa aagttggagc
gagattaaga 1440actgcaatga ccgcaatgaa gaaaatatac gcataa
14766491PRTGramella forsetii 6Met Thr Asn Tyr Phe Asn Ser Leu Ser
Leu Arg Asp Gln Leu Ala Gln 1 5 10 15 Leu Gly Thr Cys Arg Phe Met
Glu Leu Asp Glu Phe Ser Asn Glu Val 20 25 30 Ala Val Leu Lys Asp
Lys Lys Ile Val Ile Val Gly Cys Gly Ala Gln 35 40 45 Gly Leu Asn
Gln Gly Leu Asn Met Arg Asp Ser Gly Leu Asp Ile Ser 50 55 60 Tyr
Ala Leu Arg Glu Gly Ala Ile Lys Glu Lys Arg Gln Ser Trp Lys 65 70
75 80 Asn Ala Thr Glu Asn Asn Phe Asn Val Gly Thr Tyr Glu Glu Leu
Ile 85 90 95 Pro Lys Ala Asp Leu Val Ile Asn Leu Thr Pro Asp Lys
Gln His Thr 100 105 110 Ser Val Ile Lys Ala Ile Gln Pro His Ile Lys
Lys Asp Ala Val Leu 115 120 125 Ser Tyr Ser His Gly Phe Asn Ile Val
Glu Glu Gly Thr Lys Ile Arg 130 135 140 Glu Asp Ile Thr Val Ile Met
Val Ala Pro Lys Cys Pro Gly Thr Glu 145 150 155 160 Val Arg Glu Glu
Tyr Lys Arg Gly Phe Gly Val Pro Thr Leu Ile Ala 165 170 175 Val His
Pro Glu Asn Asp Pro His Gly Ile Gly Leu Asp Trp Ala Lys 180 185 190
Ala Tyr Ala Tyr Ala Thr Gly Gly His Arg Ala Gly Val Leu Glu Ser 195
200 205 Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr
Met 210 215 220 Leu Cys Gly Val Leu Gln Thr Gly Ser Ile Leu Thr Phe
Asp Lys Met 225 230 235 240 Val Ala Asp Gly Val Glu Pro Asn Tyr Ala
Ala Lys Leu Ile Gln Tyr 245 250 255 Gly Trp Glu Thr Ile Thr Glu Ala
Leu Lys His Gly Gly Ile Thr Asn 260 265 270 Met Met Asp Arg Leu Ser
Asn Pro Ala Lys Leu Arg Ala Asn Glu Ile 275 280 285 Ala Glu Glu Leu
Lys Glu Lys Met Arg Pro Leu Phe Gln Lys His Met 290 295 300 Asp Asp
Ile Ile Ser Gly Glu Phe Ser Ser Arg Met Met Arg Asp Trp 305 310 315
320 Ala Asn Asp Asp Lys Glu Leu Leu Thr Trp Arg Ala Glu Thr Glu Asn
325 330 335 Thr Ala Phe Glu Lys Thr Glu Ala Thr Ser Glu Glu Ile Lys
Glu Gln 340 345 350 Glu Tyr Phe Asp Lys Gly Val Leu Met Val Ala Phe
Val Arg Ala Gly 355 360 365 Val Glu Leu Ala Phe Glu Thr Met Val Glu
Ala Gly Ile Ile Glu Glu 370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His
Glu Thr Pro Leu Ile Ala Asn Thr 385 390 395 400 Ile Ala Arg Lys Lys
Leu Tyr Glu Met Asn Arg Val Ile Ser Asp Thr 405 410 415 Ala Glu Tyr
Gly Cys Tyr Leu Phe Asp His Ala Ala Lys Pro Leu Val 420 425 430 Lys
Asp Tyr Val Asn Ser Leu Glu Pro Glu Val Ala Gly Lys Lys Phe 435 440
445 Gly Thr Asp Cys Asn Gly Val Asp Asn Gln Lys Leu Ile His Val Asn
450 455 460 Asp Asp Leu Arg Ser His Pro Val Glu Lys Val Gly Ala Arg
Leu Arg 465 470 475 480 Thr Ala Met Thr Ala Met Lys Lys Ile Tyr Ala
485 490 71479DNACytophaga hutchinsonii 7atggcaaatt atttcaatac
tctttcatta agagaaaaat tagatcagtt aggcgtttgc 60gaattcatgg acagaagtga
gttttctgac ggtgtagctg ctttgaaagg taaaaaaatt 120gtaatcgtag
gttgtggtgc acaaggtttg aaccagggtt taaaccttcg tgattctggt
180ttagatgttt cttatacatt acgtaaagaa gccattgatt ctaaaagaca
atcattttta 240aatgcttctg aaaatggttt caaagtgggc acgtacgaag
aattaattcc tactgctgat 300ttagtaatta acttaacgcc ggataaacaa
catactgctg ttgtgtctgc agttatgcca 360ttaatgaaaa aaggttctac
cttatcttac tctcacggtt tcaacatcgt tgaagaaggt 420atgcagatcc
gtaaggacat cacggtaatc atggttgctc ctaagtctcc gggttctgaa
480gttcgtgaag aatataaaag aggtttcggt gtccctacgt tgatcgccgt
tcaccctgaa 540aacgatcctg aaggtaaagg ctgggattat gctaaggctt
actgcgtagg tacaggtggt 600gacagagctg gtgtattgaa atcatctttc
gttgctgaag taaaatctga tttaatgggt 660gagcaaacaa tcctttgtgg
tttgttgcaa actggttcta tcctttgctt cgacaaaatg 720gttgaaaaag
gcattgataa aggatatgct tctaaattga tccagtacgg
atgggaagtt 780atcacggaat cattgaaaca tggcggtatc agcggtatga
tggatcgtct ttcaaaccct 840gctaaaatca aggcgttcca ggtatctgaa
gaattgaaag atatcatgcg tccattattc 900cgtaagcatc aggatgatat
catcagcgga gaattctccc gcatcatgat ggaagactgg 960gcgaatggcg
ataaaaattt attgacatgg agagctgcaa caggtgaaac tgcatttgaa
1020aaaacgcctg caggtgacgt taaaattgct gagcaggaat attatgacaa
tggtttgctg 1080atggttgcta tggttcgtgc gggtgttgaa ctggcattcg
aaacaatgac tgaatcaggt 1140atcattgatg aatctgctta ctacgaatca
ttacacgaaa caccgcttat cgcgaacaca 1200atcgcgcgta agaaattatt
cgaaatgaac cgtgtaattt ctgatacagc tgaatacggc 1260tgctacttat
tcgatcatgc ctgtaagcca ttattggcga acttcatgaa gacagtagat
1320acagacatca ttggtaaaaa cttcaacgcg ggtaaagata atggtgttga
caaccagatg 1380ctgatcgctg taaatgaagt attacgttct cacccgatcg
aaatcgttgg tgctgaatta 1440cgtgaagcaa tgactgaaat gaaagcaatc
gtttcttaa 14798492PRTCytophaga hutchinsonii 8Met Ala Asn Tyr Phe
Asn Thr Leu Ser Leu Arg Glu Lys Leu Asp Gln 1 5 10 15 Leu Gly Val
Cys Glu Phe Met Asp Arg Ser Glu Phe Ser Asp Gly Val 20 25 30 Ala
Ala Leu Lys Gly Lys Lys Ile Val Ile Val Gly Cys Gly Ala Gln 35 40
45 Gly Leu Asn Gln Gly Leu Asn Leu Arg Asp Ser Gly Leu Asp Val Ser
50 55 60 Tyr Thr Leu Arg Lys Glu Ala Ile Asp Ser Lys Arg Gln Ser
Phe Leu 65 70 75 80 Asn Ala Ser Glu Asn Gly Phe Lys Val Gly Thr Tyr
Glu Glu Leu Ile 85 90 95 Pro Thr Ala Asp Leu Val Ile Asn Leu Thr
Pro Asp Lys Gln His Thr 100 105 110 Ala Val Val Ser Ala Val Met Pro
Leu Met Lys Lys Gly Ser Thr Leu 115 120 125 Ser Tyr Ser His Gly Phe
Asn Ile Val Glu Glu Gly Met Gln Ile Arg 130 135 140 Lys Asp Ile Thr
Val Ile Met Val Ala Pro Lys Ser Pro Gly Ser Glu 145 150 155 160 Val
Arg Glu Glu Tyr Lys Arg Gly Phe Gly Val Pro Thr Leu Ile Ala 165 170
175 Val His Pro Glu Asn Asp Pro Glu Gly Lys Gly Trp Asp Tyr Ala Lys
180 185 190 Ala Tyr Cys Val Gly Thr Gly Gly Asp Arg Ala Gly Val Leu
Lys Ser 195 200 205 Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly
Glu Gln Thr Ile 210 215 220 Leu Cys Gly Leu Leu Gln Thr Gly Ser Ile
Leu Cys Phe Asp Lys Met 225 230 235 240 Val Glu Lys Gly Ile Asp Lys
Gly Tyr Ala Ser Lys Leu Ile Gln Tyr 245 250 255 Gly Trp Glu Val Ile
Thr Glu Ser Leu Lys His Gly Gly Ile Ser Gly 260 265 270 Met Met Asp
Arg Leu Ser Asn Pro Ala Lys Ile Lys Ala Phe Gln Val 275 280 285 Ser
Glu Glu Leu Lys Asp Ile Met Arg Pro Leu Phe Arg Lys His Gln 290 295
300 Asp Asp Ile Ile Ser Gly Glu Phe Ser Arg Ile Met Met Glu Asp Trp
305 310 315 320 Ala Asn Gly Asp Lys Asn Leu Leu Thr Trp Arg Ala Ala
Thr Gly Glu 325 330 335 Thr Ala Phe Glu Lys Thr Pro Ala Gly Asp Val
Lys Ile Ala Glu Gln 340 345 350 Glu Tyr Tyr Asp Asn Gly Leu Leu Met
Val Ala Met Val Arg Ala Gly 355 360 365 Val Glu Leu Ala Phe Glu Thr
Met Thr Glu Ser Gly Ile Ile Asp Glu 370 375 380 Ser Ala Tyr Tyr Glu
Ser Leu His Glu Thr Pro Leu Ile Ala Asn Thr 385 390 395 400 Ile Ala
Arg Lys Lys Leu Phe Glu Met Asn Arg Val Ile Ser Asp Thr 405 410 415
Ala Glu Tyr Gly Cys Tyr Leu Phe Asp His Ala Cys Lys Pro Leu Leu 420
425 430 Ala Asn Phe Met Lys Thr Val Asp Thr Asp Ile Ile Gly Lys Asn
Phe 435 440 445 Asn Ala Gly Lys Asp Asn Gly Val Asp Asn Gln Met Leu
Ile Ala Val 450 455 460 Asn Glu Val Leu Arg Ser His Pro Ile Glu Ile
Val Gly Ala Glu Leu 465 470 475 480 Arg Glu Ala Met Thr Glu Met Lys
Ala Ile Val Ser 485 490 91023DNALactococcus lactis 9atggcagtta
caatgtatta tgaagatgat gtagaagtat cagcacttgc tggaaagcaa 60attgcagtaa
tcggttatgg ttcacaagga catgctcacg cacagaattt gcgtgattct
120ggtcacaacg ttatcattgg tgtgcgccac ggaaaatctt ttgataaagc
aaaagaagat 180ggctttgaaa catttgaagt aggagaagca gtagctaaag
ctgatgttat tatggttttg 240gcaccagatg aacttcaaca atccatttat
gaagaggaca tcaaaccaaa cttgaaagca 300ggttcagcac ttggttttgc
tcacggattt aatatccatt ttggctatat taaagtacca 360gaagacgttg
acgtctttat ggttgcgcct aaggctccag gtcaccttgt ccgtcggact
420tatactgaag gttttggtac accagctttg tttgtttcac accaaaatgc
aagtggtcat 480gcgcgtgaaa tcgcaatgga ttgggccaaa ggaattggtt
gtgctcgagt gggaattatt 540gaaacaactt ttaaagaaga aacagaagaa
gatttgtttg gagaacaagc tgttctatgt 600ggaggtttga cagcacttgt
tgaagccggt tttgaaacac tgacagaagc tggatacgct 660ggcgaattgg
cttactttga agttttgcac gaaatgaaat tgattgttga cctcatgtat
720gaaggtggtt ttactaaaat gcgtcaatcc atctcaaata ctgctgagtt
tggcgattat 780gtgactggtc cacggattat tactgacgaa gttaaaaaga
atatgaagct tgttttggct 840gatattcaat ctggaaaatt tgctcaagat
ttcgttgatg acttcaaagc ggggcgtcca 900aaattaatag cctatcgcga
agctgcaaaa aatcttgaaa ttgaaaaaat tggggcagag 960ctacgtcaag
caatgccatt cacacaatct ggtgatgacg atgcctttaa aatctatcag 1020taa
102310340PRTLactococcus lactis 10Met Ala Val Thr Met Tyr Tyr Glu
Asp Asp Val Glu Val Ser Ala Leu 1 5 10 15 Ala Gly Lys Gln Ile Ala
Val Ile Gly Tyr Gly Ser Gln Gly His Ala 20 25 30 His Ala Gln Asn
Leu Arg Asp Ser Gly His Asn Val Ile Ile Gly Val 35 40 45 Arg His
Gly Lys Ser Phe Asp Lys Ala Lys Glu Asp Gly Phe Glu Thr 50 55 60
Phe Glu Val Gly Glu Ala Val Ala Lys Ala Asp Val Ile Met Val Leu 65
70 75 80 Ala Pro Asp Glu Leu Gln Gln Ser Ile Tyr Glu Glu Asp Ile
Lys Pro 85 90 95 Asn Leu Lys Ala Gly Ser Ala Leu Gly Phe Ala His
Gly Phe Asn Ile 100 105 110 His Phe Gly Tyr Ile Lys Val Pro Glu Asp
Val Asp Val Phe Met Val 115 120 125 Ala Pro Lys Ala Pro Gly His Leu
Val Arg Arg Thr Tyr Thr Glu Gly 130 135 140 Phe Gly Thr Pro Ala Leu
Phe Val Ser His Gln Asn Ala Ser Gly His 145 150 155 160 Ala Arg Glu
Ile Ala Met Asp Trp Ala Lys Gly Ile Gly Cys Ala Arg 165 170 175 Val
Gly Ile Ile Glu Thr Thr Phe Lys Glu Glu Thr Glu Glu Asp Leu 180 185
190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Thr Ala Leu Val Glu
195 200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Ala Gly Glu
Leu Ala 210 215 220 Tyr Phe Glu Val Leu His Glu Met Lys Leu Ile Val
Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Phe Thr Lys Met Arg Gln
Ser Ile Ser Asn Thr Ala Glu 245 250 255 Phe Gly Asp Tyr Val Thr Gly
Pro Arg Ile Ile Thr Asp Glu Val Lys 260 265 270 Lys Asn Met Lys Leu
Val Leu Ala Asp Ile Gln Ser Gly Lys Phe Ala 275 280 285 Gln Asp Phe
Val Asp Asp Phe Lys Ala Gly Arg Pro Lys Leu Ile Ala 290 295 300 Tyr
Arg Glu Ala Ala Lys Asn Leu Glu Ile Glu Lys Ile Gly Ala Glu 305 310
315 320 Leu Arg Gln Ala Met Pro Phe Thr Gln Ser Gly Asp Asp Asp Ala
Phe 325 330 335 Lys Ile Tyr Gln 340 111023DNALactococcus lactis
11atggcagtta caatgtatta tgaagatgat gtagaagtat cagcacttgc tggaaagcaa
60attgcagtaa tcggttatgg ttcacaagga catgctcacg cacagaattt gcgtgattct
120ggtcacaacg ttatcattgg tgtgcgccac ggaaaatctt ttgataaagc
aaaagaagat 180ggctttgaaa catttgaagt aggagaagcg gtagctaaag
ctgatgttat tatggttttg 240gcgccagatg aacttcaaca atccatttat
gaagaggaca tcaaaccaaa cttgaaagca 300ggttcagcac ttggttttgc
tcacggattt aatatccatt ttggctatat taaagtacca 360gaagacgttg
acgtctttat ggttgcacct aaggctccag gtcaccttgt ccgtcggact
420tatactgaag gttttggtac accagctttg tttgtttcac accaaaatgc
aagtggtcat 480gcgcgtgaaa tcgcaatgga ttgggccaaa ggaattggtt
gtgctcgagt gggaattatt 540gaaacaacct ttaaagaaga aacagaagaa
gatttgtttg gagaacaagc tgttctatgt 600ggaggtttga cagcacttgt
tgaagccggt tttgaaacac tgacagaagc tggatacgct 660ggcgaattgg
cttactttga agttttgcac gaaatgaaat tgattgttga cctcatgtat
720gaaggtggtt ttactaaaat gcgtcaatcc atctcaaata ctgctgagtt
tggcgattat 780gtgactggtc caaggattat tactgacgca gttaaaaaga
atatgaagct tgttttggct 840gatattcaat ctggaaaatt tgctcaagat
ttcgttgatg acttcaaagc ggggcgtcca 900aaattaacag cctatcgcga
agctgctaaa aatcttgaaa ttgaaaaaat tggggcagaa 960ttacgtaaag
caatgccatt cacacaatct ggtgatgacg atgcctttaa aatctatcag 1020taa
102312340PRTLactococcus lactis 12Met Ala Val Thr Met Tyr Tyr Glu
Asp Asp Val Glu Val Ser Ala Leu 1 5 10 15 Ala Gly Lys Gln Ile Ala
Val Ile Gly Tyr Gly Ser Gln Gly His Ala 20 25 30 His Ala Gln Asn
Leu Arg Asp Ser Gly His Asn Val Ile Ile Gly Val 35 40 45 Arg His
Gly Lys Ser Phe Asp Lys Ala Lys Glu Asp Gly Phe Glu Thr 50 55 60
Phe Glu Val Gly Glu Ala Val Ala Lys Ala Asp Val Ile Met Val Leu 65
70 75 80 Ala Pro Asp Glu Leu Gln Gln Ser Ile Tyr Glu Glu Asp Ile
Lys Pro 85 90 95 Asn Leu Lys Ala Gly Ser Ala Leu Gly Phe Ala His
Gly Phe Asn Ile 100 105 110 His Phe Gly Tyr Ile Lys Val Pro Glu Asp
Val Asp Val Phe Met Val 115 120 125 Ala Pro Lys Ala Pro Gly His Leu
Val Arg Arg Thr Tyr Thr Glu Gly 130 135 140 Phe Gly Thr Pro Ala Leu
Phe Val Ser His Gln Asn Ala Ser Gly His 145 150 155 160 Ala Arg Glu
Ile Ala Met Asp Trp Ala Lys Gly Ile Gly Cys Ala Arg 165 170 175 Val
Gly Ile Ile Glu Thr Thr Phe Lys Glu Glu Thr Glu Glu Asp Leu 180 185
190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Thr Ala Leu Val Glu
195 200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Ala Gly Glu
Leu Ala 210 215 220 Tyr Phe Glu Val Leu His Glu Met Lys Leu Ile Val
Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Phe Thr Lys Met Arg Gln
Ser Ile Ser Asn Thr Ala Glu 245 250 255 Phe Gly Asp Tyr Val Thr Gly
Pro Arg Ile Ile Thr Asp Ala Val Lys 260 265 270 Lys Asn Met Lys Leu
Val Leu Ala Asp Ile Gln Ser Gly Lys Phe Ala 275 280 285 Gln Asp Phe
Val Asp Asp Phe Lys Ala Gly Arg Pro Lys Leu Thr Ala 290 295 300 Tyr
Arg Glu Ala Ala Lys Asn Leu Glu Ile Glu Lys Ile Gly Ala Glu 305 310
315 320 Leu Arg Lys Ala Met Pro Phe Thr Gln Ser Gly Asp Asp Asp Ala
Phe 325 330 335 Lys Ile Tyr Gln 340 131023DNALactococcus lactis
13atggcagtta caatgtatta tgaagatgat gtagaagtat cagcacttgc tggaaagcaa
60attgcagtaa tcggttatgg ttcacaagga catgctcacg cacagaattt gcatgattct
120ggtcacaacg ttatcattgg tgtgcgccac ggaaaatctt ttgataaagc
aaaagaagat 180ggctttgaaa catttgaagt aggagaagcg gtagctaaag
ctgatgttat tatggttttg 240gcgccagatg aacttcaaca atccatttat
gaagaggaca tcaaaccaaa cttgaaagca 300ggttcagcac ttggttttgc
tcacggattt aatatccatt ttggctatat taaagtacca 360gaagacgttg
acgtctttat ggttgcacct aaggctccag gtcaccttgt ccgtcggact
420tatactgaag gttttggtac accagctttg tttgtttcac accaaaatgc
aagtggtcat 480gcgcgtgaaa tcgcaatgga ttgggccaaa ggaattggtt
gtgctcgagt gggaattatt 540gaaacaacct ttaaagaaga aacagaagaa
gatttgtttg gagaacaagc tgttctatgt 600ggaggtttga cagcacttgt
tgaagccggt tttgaaacac tgacagaagc tggatacgct 660ggcgaattgg
cttactttga agttttgcac gaaatgaaat tgattgttga cctcatgtat
720gaaggtggtt ttactaaaat gcgtcaatcc atctcaaata ctgctgagtt
tggcgattat 780gtgactggtc caaggattat tactgacgca gttaaaaaga
atatgaagct tgttttggct 840gatattcaat ctggaaaatt tgctcaagat
ttcgttgatg acttcaaagc ggggcgtcca 900aaattaacag cctatcgcga
agctgctaaa aatcttgaaa ttgaaaaaat tggggcagaa 960ttacgtaaag
caatgccatt cacacaatct ggtgatgacg atgcctttaa aatctatcag 1020taa
102314340PRTLactococcus lactis 14Met Ala Val Thr Met Tyr Tyr Glu
Asp Asp Val Glu Val Ser Ala Leu 1 5 10 15 Ala Gly Lys Gln Ile Ala
Val Ile Gly Tyr Gly Ser Gln Gly His Ala 20 25 30 His Ala Gln Asn
Leu His Asp Ser Gly His Asn Val Ile Ile Gly Val 35 40 45 Arg His
Gly Lys Ser Phe Asp Lys Ala Lys Glu Asp Gly Phe Glu Thr 50 55 60
Phe Glu Val Gly Glu Ala Val Ala Lys Ala Asp Val Ile Met Val Leu 65
70 75 80 Ala Pro Asp Glu Leu Gln Gln Ser Ile Tyr Glu Glu Asp Ile
Lys Pro 85 90 95 Asn Leu Lys Ala Gly Ser Ala Leu Gly Phe Ala His
Gly Phe Asn Ile 100 105 110 His Phe Gly Tyr Ile Lys Val Pro Glu Asp
Val Asp Val Phe Met Val 115 120 125 Ala Pro Lys Ala Pro Gly His Leu
Val Arg Arg Thr Tyr Thr Glu Gly 130 135 140 Phe Gly Thr Pro Ala Leu
Phe Val Ser His Gln Asn Ala Ser Gly His 145 150 155 160 Ala Arg Glu
Ile Ala Met Asp Trp Ala Lys Gly Ile Gly Cys Ala Arg 165 170 175 Val
Gly Ile Ile Glu Thr Thr Phe Lys Glu Glu Thr Glu Glu Asp Leu 180 185
190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Thr Ala Leu Val Glu
195 200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Ala Gly Glu
Leu Ala 210 215 220 Tyr Phe Glu Val Leu His Glu Met Lys Leu Ile Val
Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Phe Thr Lys Met Arg Gln
Ser Ile Ser Asn Thr Ala Glu 245 250 255 Phe Gly Asp Tyr Val Thr Gly
Pro Arg Ile Ile Thr Asp Ala Val Lys 260 265 270 Lys Asn Met Lys Leu
Val Leu Ala Asp Ile Gln Ser Gly Lys Phe Ala 275 280 285 Gln Asp Phe
Val Asp Asp Phe Lys Ala Gly Arg Pro Lys Leu Thr Ala 290 295 300 Tyr
Arg Glu Ala Ala Lys Asn Leu Glu Ile Glu Lys Ile Gly Ala Glu 305 310
315 320 Leu Arg Lys Ala Met Pro Phe Thr Gln Ser Gly Asp Asp Asp Ala
Phe 325 330 335 Lys Ile Tyr Gln 340 151023DNALactococcus lactis
15atggcagtta caatgtatta tgaagaagat gtagaagtag ccgcactcgc gggtaagaaa
60atcgcagtga ttggatatgg ctcacaagga cacgctcatg cacaaaactt gcgtgattct
120ggtcatgatg tgattattgg tgtccgtcag gggaaatctt ttgataaagc
aaaagaagat 180ggttttgaaa catttgaagt aggagaagca gtagctaaag
ctgacgtcat tatggttctg 240gcacctgatg aacttcaaca atctatttat
gaagaggaca taaaaccaaa tttgaaagca 300ggttcagcac ttggttttgc
ccatggtttc aatattcatt ttggctatat tgaagttcca 360gaagatgttg
atgtcttcat ggttgcgcca aaagcgccgg gacatctcgt tcggcggact
420tttaccgaag gtttcggaac gccagctttg ttcgtttcgc atcaaaatgc
cactggtcat 480gcgcgtgaaa ttgccatgga ctgggccaaa ggaattggct
gtgcccgtgt cggtatcatt 540gaaacaactt tcaaagaaga aacagaagaa
gatttgtttg gcgaacaggc cgtgctttgt 600ggcggtttga cagcacttgt
tgaagctggt tttgaaacac tgacagaagc tggatatgct 660ggcgaattgg
cttactttga agtgctgcat gaaatgaaat tgattgttga ccttatgtac
720gaaggtggtt tcactaaaat gcgtcagtca atctcaaaca ctgccgaatt
tggtgattat 780gtgactggac cacgcattat tactgacgaa gttaaaaaga
atatgaaact cgtgttggct 840gacattcaat caggaaaatt tgcgcaagat
ttcgttgatg atttcaaagc tggacgtcca 900aaattaactg cttatcgtga
agcagctaaa aatctggaaa ttgaaaaaat cggtgcagaa 960ctacgtaaag
caatgccatt tacacaatct ggtgatgacg acgcctttaa aatttatcaa
1020taa 102316340PRTLactococcus lactis 16Met Ala Val Thr Met Tyr
Tyr Glu Glu Asp Val Glu Val Ala Ala Leu 1 5 10 15 Ala Gly Lys Lys
Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly His Ala 20 25 30 His Ala
Gln Asn Leu Arg Asp Ser Gly His Asp Val Ile Ile Gly Val 35 40 45
Arg Gln Gly Lys Ser Phe Asp Lys Ala Lys Glu Asp Gly Phe Glu Thr 50
55 60 Phe Glu Val Gly Glu Ala Val Ala Lys Ala Asp Val Ile Met Val
Leu 65 70 75 80 Ala Pro Asp Glu Leu Gln Gln Ser Ile Tyr Glu Glu Asp
Ile Lys Pro 85 90 95 Asn Leu Lys Ala Gly Ser Ala Leu Gly Phe Ala
His Gly Phe Asn Ile 100 105 110 His Phe Gly Tyr Ile Glu Val Pro Glu
Asp Val Asp Val Phe Met Val 115 120 125 Ala Pro Lys Ala Pro Gly His
Leu Val Arg Arg Thr Phe Thr Glu Gly 130 135 140 Phe Gly Thr Pro Ala
Leu Phe Val Ser His Gln Asn Ala Thr Gly His 145 150 155 160 Ala Arg
Glu Ile Ala Met Asp Trp Ala Lys Gly Ile Gly Cys Ala Arg 165 170 175
Val Gly Ile Ile Glu Thr Thr Phe Lys Glu Glu Thr Glu Glu Asp Leu 180
185 190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Thr Ala Leu Val
Glu 195 200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Ala Gly
Glu Leu Ala 210 215 220 Tyr Phe Glu Val Leu His Glu Met Lys Leu Ile
Val Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Phe Thr Lys Met Arg
Gln Ser Ile Ser Asn Thr Ala Glu 245 250 255 Phe Gly Asp Tyr Val Thr
Gly Pro Arg Ile Ile Thr Asp Glu Val Lys 260 265 270 Lys Asn Met Lys
Leu Val Leu Ala Asp Ile Gln Ser Gly Lys Phe Ala 275 280 285 Gln Asp
Phe Val Asp Asp Phe Lys Ala Gly Arg Pro Lys Leu Thr Ala 290 295 300
Tyr Arg Glu Ala Ala Lys Asn Leu Glu Ile Glu Lys Ile Gly Ala Glu 305
310 315 320 Leu Arg Lys Ala Met Pro Phe Thr Gln Ser Gly Asp Asp Asp
Ala Phe 325 330 335 Lys Ile Tyr Gln 340 171023DNALactococcus lactis
17atggcagtta caatgtatta tgaagaagat gtagaagtag ccgcactcgc gggtaagaaa
60atcgcagtga ttggatatgg ctcacaagga cacgctcatg cacaaaactt gcgtgattct
120ggtcatgatg tgattattgg tgtccgtcag gggaaatctt ttgataaagc
aaaagaagat 180ggttttgaaa catttgaagt aggagaagca gtagctaaag
ctgacgtcat tatggttctg 240gcacctgatg aacttcaaca atctatttat
gaagaggaca taaaaccaaa tttgaaagca 300ggttcagcac ttggttttgc
ccatggtttc aatattcatt ttggctatat tgaagttcca 360gaagatgttg
atgtcttcat ggttgcgcca aaagcgccgg gacatctcgt tcggcggact
420tttaccgaag gtttcggaac gccagctttg ttcgtttcgc atcaaaatgc
cactggtcat 480gcgcgtgaaa ttgccatgga ctgggccaaa ggaattggct
gtgcccgtgt cggtatcatt 540gaaacaactt tcaaagaaga aacagaagaa
gatttgtttg gcgaacaggc cgtgctttgt 600ggcggtttga cagcacttgt
tgaagctggt tttgaaacac tgacagaagc tggatatgct 660ggcgaattgg
cttactttga agtgctgcat gaaatgaaat tgattgttga ccttatgtac
720gaaggtggtt tcactaaaat gcgtcagtca atctcaaaca ctgccgaatt
tggtgattat 780gtgactggac cacgcattat tactgacgaa gttaaaaaga
atatgaaact cgtgttggct 840gacattcaat caggaaaatt tgcgcaagat
ttcgttgatg atttcaaagc tggacgtcca 900aaattaactg cttatcgtga
agcagctaaa aatctggaaa ttgaaaaaat cggtgcagaa 960ctacgtaaag
caatgccatt tacacaatct ggtgatgacg acgcctttaa aatttatcaa 1020taa
102318340PRTLactococcus lactis 18Met Ala Val Thr Met Tyr Tyr Glu
Glu Asp Val Glu Val Ala Ala Leu 1 5 10 15 Ala Gly Lys Lys Ile Ala
Val Ile Gly Tyr Gly Ser Gln Gly His Ala 20 25 30 His Ala Gln Asn
Leu Arg Asp Ser Gly His Asp Val Ile Ile Gly Val 35 40 45 Arg Gln
Gly Lys Ser Phe Asp Lys Ala Lys Glu Asp Gly Phe Glu Thr 50 55 60
Phe Glu Val Gly Glu Ala Val Ala Lys Ala Asp Val Ile Met Val Leu 65
70 75 80 Ala Pro Asp Glu Leu Gln Gln Ser Ile Tyr Glu Glu Asp Ile
Lys Pro 85 90 95 Asn Leu Lys Ala Gly Ser Ala Leu Gly Phe Ala His
Gly Phe Asn Ile 100 105 110 His Phe Gly Tyr Ile Glu Val Pro Glu Asp
Val Asp Val Phe Met Val 115 120 125 Ala Pro Lys Ala Pro Gly His Leu
Val Arg Arg Thr Phe Thr Glu Gly 130 135 140 Phe Gly Thr Pro Ala Leu
Phe Val Ser His Gln Asn Ala Thr Gly His 145 150 155 160 Ala Arg Glu
Ile Ala Met Asp Trp Ala Lys Gly Ile Gly Cys Ala Arg 165 170 175 Val
Gly Ile Ile Glu Thr Thr Phe Lys Glu Glu Thr Glu Glu Asp Leu 180 185
190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Thr Ala Leu Val Glu
195 200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Ala Gly Glu
Leu Ala 210 215 220 Tyr Phe Glu Val Leu His Glu Met Lys Leu Ile Val
Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Phe Thr Lys Met Arg Gln
Ser Ile Ser Asn Thr Ala Glu 245 250 255 Phe Gly Asp Tyr Val Thr Gly
Pro Arg Ile Ile Thr Asp Glu Val Lys 260 265 270 Lys Asn Met Lys Leu
Val Leu Ala Asp Ile Gln Ser Gly Lys Phe Ala 275 280 285 Gln Asp Phe
Val Asp Asp Phe Lys Ala Gly Arg Pro Lys Leu Thr Ala 290 295 300 Tyr
Arg Glu Ala Ala Lys Asn Leu Glu Ile Glu Lys Ile Gly Ala Glu 305 310
315 320 Leu Arg Lys Ala Met Pro Phe Thr Gln Ser Gly Asp Asp Asp Ala
Phe 325 330 335 Lys Ile Tyr Gln 340 191023DNALactococcus lactis
19atggcagtta caatgtatta tgaagaagat gtagaagtag ccgcactcgc gggtaagaaa
60atcgcagtga ttggatatgg ctcacaagga cacgctcatg cacaaaactt gcgtgattct
120ggtcatgatg tgattattgg cgttcgtcag gggaaatctt ttgatagagc
aaaagaagat 180ggctttgaaa catttgaagt aggagaagca gtagctaaag
ctgatgtcat tatggttctg 240gcacctgatg aacttcaaca atctatttat
gaagaggaca taaaaccaaa tttgaaatca 300ggttcagcac ttggttttgc
ccatggtttc aatattcatt ttggctatat tgaagttcca 360gaagatgttg
atgtcttcat ggttgcgcca aaagcgccgg gacatctcgt tcggcggact
420tttaccgaag gtttcggaac gccagctttg ttcgtttcgc atcaaaatgc
cactggtcat 480gcgcgtgaaa tcgctatgga ctgggcgaaa ggcattggtt
gtgcccgtgt gggaattatc 540gaaacaactt tcaaagaaga aacagaagaa
gatttgtttg gcgaacaagc tgtgctttgt 600ggtggtttga cagcacttgt
tgaagctggt tttgaaacac tgacagaagc tagatatgct 660ggtgaattgg
cttactttga agtgctgcat gaaatgaaat tgattgttga ccttatgtac
720gaaggtggtt tcactaaaat gcgtcagtca atctcaaata ctgccgaatt
tggcgattat 780gtgactggac cacgcattat tactgacgaa gttaaaaaga
atatgaaact cgtgttggct 840gacattcaat caggaaaatt tgcgcaagat
ttcgttgatg atttcaaagc tggacgtcca 900aaattaactg cttatcgtga
agcagctaaa aatctggaaa ttgaaaaaat cggtgcagaa 960ctacgtaaag
caatgccatt tacacaatct ggtgatgacg acgcctttaa aatttatcaa 1020taa
102320340PRTLactococcus lactis 20Met Ala Val Thr Met Tyr Tyr Glu
Glu Asp Val Glu Val Ala Ala Leu 1 5 10 15 Ala Gly Lys Lys Ile Ala
Val Ile Gly Tyr Gly Ser Gln Gly His Ala 20 25 30 His Ala Gln Asn
Leu Arg Asp Ser Gly His Asp Val Ile Ile Gly Val 35 40 45 Arg Gln
Gly Lys Ser Phe Asp Arg Ala Lys Glu Asp Gly Phe Glu Thr 50 55 60
Phe Glu Val Gly Glu Ala Val Ala Lys Ala Asp Val Ile Met Val Leu 65
70 75 80 Ala Pro Asp Glu Leu Gln Gln Ser Ile Tyr Glu Glu Asp Ile
Lys Pro 85 90 95 Asn Leu Lys Ser Gly Ser Ala Leu Gly Phe Ala His
Gly Phe Asn Ile 100 105 110 His Phe Gly Tyr Ile Glu Val Pro Glu Asp
Val Asp Val Phe Met Val 115 120 125 Ala Pro Lys Ala Pro Gly His Leu
Val Arg Arg Thr Phe Thr Glu Gly 130 135 140 Phe Gly Thr Pro Ala Leu
Phe Val Ser His Gln Asn Ala Thr Gly His 145 150 155 160 Ala Arg Glu
Ile Ala Met Asp Trp Ala Lys Gly Ile Gly Cys Ala Arg 165 170 175 Val
Gly Ile Ile Glu Thr Thr Phe Lys Glu Glu Thr Glu Glu Asp Leu 180 185
190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Thr Ala Leu Val Glu
195 200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Arg Tyr Ala Gly Glu
Leu Ala 210 215 220 Tyr Phe Glu Val Leu His Glu Met Lys Leu Ile Val
Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Phe Thr Lys Met Arg Gln
Ser Ile Ser Asn Thr Ala Glu 245 250 255 Phe Gly Asp Tyr Val Thr Gly
Pro Arg Ile Ile Thr Asp Glu Val Lys 260 265 270 Lys Asn Met Lys Leu
Val Leu Ala Asp Ile Gln Ser Gly Lys Phe Ala 275 280 285 Gln Asp Phe
Val Asp Asp Phe Lys Ala Gly Arg Pro Lys Leu Thr Ala 290 295 300 Tyr
Arg Glu Ala Ala Lys Asn Leu Glu Ile Glu Lys Ile Gly Ala Glu 305 310
315 320 Leu Arg Lys Ala Met Pro Phe Thr Gln Ser Gly Asp Asp Asp Ala
Phe 325 330 335 Lys Ile Tyr Gln 340 211034DNALactococcus lactis
21atggcagtta caatgtatta tgaagatgat gtagaagtat cagcacttgc tggaaagcaa
60attgcagtaa tcggttatgg ttcacaagga catgctcacg cacagaattt gcgtgattct
120ggtcacaacg ttatcattgg tgtgcgccac ggaaaatctt ttgataaagc
aaaagaagat 180ggctttgaaa catttgaagt aggagaagca gtagctaaag
ctgatgttat tatggttttg 240gcaccagatg aacttcaaca atccatttat
gaagaggaca tcaaaccaaa cttgaaagca 300ggttcagcac ttggttttgc
tcacggattt aatatccatt ttggctatat taaagtacca 360gaagacgttg
acgtctttat ggttgcgcct aaggctccag gtcaccttgt ccgtcggact
420tatactgaag gttttggtac accagctttg tttgtttcac accaaaatgc
aagtggtcat 480gcgcgtgaaa tcgcaatgga ttgggccaaa ggaattggtt
gtgctcgagt gggaattatt 540gaaacaactt ttaaagaaga aacagaagaa
gatttgtttg gagaacaagc tgttctatgt 600ggaggtttga cagcacttgt
tgaagccggt tttgaaacac tgacagaagc tggatacgct 660ggcgaattgg
cttactttga agttttgcac gaaatgaaat tgattgttga cctcatgtat
720gaaggtggtt ttactaaaat gcgtcaatcc atctcaaata ctgctgagtt
tggcgattat 780gtgactggtc cacggattat tactgacgaa gttaaaaaga
atatgaagct tgttttggct 840gatattcaat ctggaaaatt tgctcaagat
ttcgttgatg acttcaaagc ggggcgtcca 900aaattaatag cctatcgcga
agctgcaaaa aatcttgaaa ttgaaaaaat tggggcagag 960cacgtcaagc
aatgccattc acacaatctg gtgatgacga tgcctttaaa atctatcagt
1020aatttctctt attg 103422344PRTLactococcus lactis 22Met Ala Val
Thr Met Tyr Tyr Glu Asp Asp Val Glu Val Ser Ala Leu 1 5 10 15 Ala
Gly Lys Gln Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly His Ala 20 25
30 His Ala Gln Asn Leu Arg Asp Ser Gly His Asn Val Ile Ile Gly Val
35 40 45 Arg His Gly Lys Ser Phe Asp Lys Ala Lys Glu Asp Gly Phe
Glu Thr 50 55 60 Phe Glu Val Gly Glu Ala Val Ala Lys Ala Asp Val
Ile Met Val Leu 65 70 75 80 Ala Pro Asp Glu Leu Gln Gln Ser Ile Tyr
Glu Glu Asp Ile Lys Pro 85 90 95 Asn Leu Lys Ala Gly Ser Ala Leu
Gly Phe Ala His Gly Phe Asn Ile 100 105 110 His Phe Gly Tyr Ile Lys
Val Pro Glu Asp Val Asp Val Phe Met Val 115 120 125 Ala Pro Lys Ala
Pro Gly His Leu Val Arg Arg Thr Tyr Thr Glu Gly 130 135 140 Phe Gly
Thr Pro Ala Leu Phe Val Ser His Gln Asn Ala Ser Gly His 145 150 155
160 Ala Arg Glu Ile Ala Met Asp Trp Ala Lys Gly Ile Gly Cys Ala Arg
165 170 175 Val Gly Ile Ile Glu Thr Thr Phe Lys Glu Glu Thr Glu Glu
Asp Leu 180 185 190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Thr
Ala Leu Val Glu 195 200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Gly
Tyr Ala Gly Glu Leu Ala 210 215 220 Tyr Phe Glu Val Leu His Glu Met
Lys Leu Ile Val Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Phe Thr
Lys Met Arg Gln Ser Ile Ser Asn Thr Ala Glu 245 250 255 Phe Gly Asp
Tyr Val Thr Gly Pro Arg Ile Ile Thr Asp Glu Val Lys 260 265 270 Lys
Asn Met Lys Leu Val Leu Ala Asp Ile Gln Ser Gly Lys Phe Ala 275 280
285 Gln Asp Phe Val Asp Asp Phe Lys Ala Gly Arg Pro Lys Leu Ile Ala
290 295 300 Tyr Arg Glu Ala Ala Lys Asn Leu Glu Ile Glu Lys Ile Gly
Ala Glu 305 310 315 320 His Val Lys Gln Cys His Ser His Asn Leu Val
Met Thr Met Pro Leu 325 330 335 Lys Ser Ile Ser Asn Phe Ser Tyr 340
231023DNAStreptococcus equinus 23atggcagtaa caatggaata cgaaaaagat
gtaaaagtag cagctcttga tggtaaaaaa 60attgccgtta tcggttatgg atcacaaggt
catgctcatg ctcaaaactt acgtgattca 120ggtcacgatg ttatcattgg
ggttcgccat ggtaaatcat tcgacaaagc aaaagaagat 180ggtttcgaaa
catatgaagt agctgaagca acaaaacttg ctgatgttat catggttttg
240gcaccagatg aaatccaagc aaaactttat gctgaagaaa ttgcaccaaa
tcttgaagca 300ggtaatgcac ttggttttgc tcatggtttc aatatccgtt
ttgaatatat taaagctcca 360gaaacagtag atgtctttat gtgtgcgcct
aaaggtccag gtcaccttgt acgccgtact 420tacactgaag gatttggtgt
gccagcactt tacgctgttt atcaagatgc tactggtcat 480gctaaagaca
tcgcaatgga ctggtctaaa ggtatcggtg ctgcgcgtgt tggacttctt
540gaaactacat tcaaagaaga aactgaagaa gatttgttcg gtgaacaagc
agttctttgt 600ggtggtttga ctgcccttat cgaagcaggt tttgaagttc
ttactgaagc aggctatgct 660ccagaattgg cttacttcga agttcttcat
gaaatgaaac ttatcgttga ccttatttac 720gaaggtggat tcaagaaaat
gcgtcaatca atttcaaata cagctgaatt tggtgactat 780gtttcaggtc
cacgtgtcat cactaaagat gttaaagaaa atatgaaagc cgttcttgct
840gatattcaat caggtaaatt tgctgaagaa tttgtaagcg actataaagc
tggtcgtcca 900aaacttgaag cttatcgtaa agaagctgca gaacttgaaa
ttgaaaaagt gggtgcagaa 960cttcgtaaag caatgccttt tgttaaccaa
aatgatgacg atgcattcaa aatttataac 1020taa 102324340PRTStreptococcus
equinus 24Met Ala Val Thr Met Glu Tyr Glu Lys Asp Val Lys Val Ala
Ala Leu 1 5 10 15 Asp Gly Lys Lys Ile Ala Val Ile Gly Tyr Gly Ser
Gln Gly His Ala 20 25 30 His Ala Gln Asn Leu Arg Asp Ser Gly His
Asp Val Ile Ile Gly Val 35 40 45 Arg His Gly Lys Ser Phe Asp Lys
Ala Lys Glu Asp Gly Phe Glu Thr 50 55 60 Tyr Glu Val Ala Glu Ala
Thr Lys Leu Ala Asp Val Ile Met Val Leu 65 70 75 80 Ala Pro Asp Glu
Ile Gln Ala Lys Leu Tyr Ala Glu Glu Ile Ala Pro 85 90 95 Asn Leu
Glu Ala Gly Asn Ala Leu Gly Phe Ala His Gly Phe Asn Ile 100 105 110
Arg Phe Glu Tyr Ile Lys Ala Pro Glu Thr Val Asp Val Phe Met Cys 115
120 125 Ala Pro Lys Gly Pro Gly His Leu Val Arg Arg Thr Tyr Thr Glu
Gly 130 135 140 Phe Gly Val Pro Ala Leu Tyr Ala Val Tyr Gln Asp Ala
Thr Gly His 145 150 155 160 Ala Lys Asp Ile Ala Met Asp Trp Ser Lys
Gly Ile Gly Ala Ala Arg 165 170 175 Val Gly Leu Leu Glu Thr Thr Phe
Lys Glu Glu Thr Glu Glu Asp Leu 180 185 190 Phe Gly Glu Gln Ala Val
Leu Cys Gly Gly Leu Thr Ala Leu Ile Glu 195 200 205 Ala Gly Phe Glu
Val Leu Thr Glu Ala Gly Tyr Ala Pro Glu Leu Ala 210 215 220 Tyr Phe
Glu Val Leu His Glu Met Lys Leu Ile Val
Asp Leu Ile Tyr 225 230 235 240 Glu Gly Gly Phe Lys Lys Met Arg Gln
Ser Ile Ser Asn Thr Ala Glu 245 250 255 Phe Gly Asp Tyr Val Ser Gly
Pro Arg Val Ile Thr Lys Asp Val Lys 260 265 270 Glu Asn Met Lys Ala
Val Leu Ala Asp Ile Gln Ser Gly Lys Phe Ala 275 280 285 Glu Glu Phe
Val Ser Asp Tyr Lys Ala Gly Arg Pro Lys Leu Glu Ala 290 295 300 Tyr
Arg Lys Glu Ala Ala Glu Leu Glu Ile Glu Lys Val Gly Ala Glu 305 310
315 320 Leu Arg Lys Ala Met Pro Phe Val Asn Gln Asn Asp Asp Asp Ala
Phe 325 330 335 Lys Ile Tyr Asn 340 251023DNAStreptococcus
infantarius 25atggcagtaa caatggaata cgaaaaagac gtaaaagtag
cagctcttga tggtaaaaaa 60attgccgtta ttggttatgg atcacaaggt catgctcatg
ctcaaaactt gcgtgactca 120ggtcacgatg ttatcattgg ggttcgccat
ggtaaatcat tcgataaagc aaaagaagat 180ggatttgata cttatgaagt
agcagaagca acaaaacttg ctgatgttat catggtattg 240gctcctgatg
aaatccaagc taaactttat gctgaagaaa tcgctccaaa ccttgaagct
300ggtaacgctc ttggatttgc acatggtttt aatatccgtt ttggatacat
taaagctcca 360gaaacagtag atgtcttcat gtgtgctcct aaaggaccag
gtcaccttgt tcgtcgtact 420tacacagaag gatttggtgt accagcactt
tacgctgttt accaagatgc tactggtaat 480gctaaagaca tcgcaatgga
ttggtctaaa ggtatcggtg ctgcacgtgt tggacttctt 540gaaacaacat
ttaaagaaga aactgaagaa gacctctttg gtgaacaagc agtactttgt
600ggtggtttaa ctgctcttat cgaagctggt tttgaagttc ttactgaagc
tggctatgct 660ccagaattgg cttactttga agttcttcat gaaatgaaac
ttatcgttga ccttatctac 720gaaggtggat tcaagaaaat gcgtcaatca
atttcaaata cagctgaatt tggtgactac 780gtatctggac cacgtgttat
cactaaagat gttaaagaaa atatgaaagc tgttcttgct 840gatatccaat
caggtaaatt cgctgaagat tttgttaacg actaccaagc aggtcgtcca
900aaacttgaag cataccgtaa agaagctgca gctcttgaaa ttgaaaaagt
gggtgctgaa 960cttcgtaaag caatgccttt tgttaaccaa aacgatgacg
atgcattcaa aatttataac 1020taa 102326340PRTStreptococcus infantarius
26Met Ala Val Thr Met Glu Tyr Glu Lys Asp Val Lys Val Ala Ala Leu 1
5 10 15 Asp Gly Lys Lys Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly His
Ala 20 25 30 His Ala Gln Asn Leu Arg Asp Ser Gly His Asp Val Ile
Ile Gly Val 35 40 45 Arg His Gly Lys Ser Phe Asp Lys Ala Lys Glu
Asp Gly Phe Asp Thr 50 55 60 Tyr Glu Val Ala Glu Ala Thr Lys Leu
Ala Asp Val Ile Met Val Leu 65 70 75 80 Ala Pro Asp Glu Ile Gln Ala
Lys Leu Tyr Ala Glu Glu Ile Ala Pro 85 90 95 Asn Leu Glu Ala Gly
Asn Ala Leu Gly Phe Ala His Gly Phe Asn Ile 100 105 110 Arg Phe Gly
Tyr Ile Lys Ala Pro Glu Thr Val Asp Val Phe Met Cys 115 120 125 Ala
Pro Lys Gly Pro Gly His Leu Val Arg Arg Thr Tyr Thr Glu Gly 130 135
140 Phe Gly Val Pro Ala Leu Tyr Ala Val Tyr Gln Asp Ala Thr Gly Asn
145 150 155 160 Ala Lys Asp Ile Ala Met Asp Trp Ser Lys Gly Ile Gly
Ala Ala Arg 165 170 175 Val Gly Leu Leu Glu Thr Thr Phe Lys Glu Glu
Thr Glu Glu Asp Leu 180 185 190 Phe Gly Glu Gln Ala Val Leu Cys Gly
Gly Leu Thr Ala Leu Ile Glu 195 200 205 Ala Gly Phe Glu Val Leu Thr
Glu Ala Gly Tyr Ala Pro Glu Leu Ala 210 215 220 Tyr Phe Glu Val Leu
His Glu Met Lys Leu Ile Val Asp Leu Ile Tyr 225 230 235 240 Glu Gly
Gly Phe Lys Lys Met Arg Gln Ser Ile Ser Asn Thr Ala Glu 245 250 255
Phe Gly Asp Tyr Val Ser Gly Pro Arg Val Ile Thr Lys Asp Val Lys 260
265 270 Glu Asn Met Lys Ala Val Leu Ala Asp Ile Gln Ser Gly Lys Phe
Ala 275 280 285 Glu Asp Phe Val Asn Asp Tyr Gln Ala Gly Arg Pro Lys
Leu Glu Ala 290 295 300 Tyr Arg Lys Glu Ala Ala Ala Leu Glu Ile Glu
Lys Val Gly Ala Glu 305 310 315 320 Leu Arg Lys Ala Met Pro Phe Val
Asn Gln Asn Asp Asp Asp Ala Phe 325 330 335 Lys Ile Tyr Asn 340
27993DNAMethanococcus maripaludis 27atgaaggtat tctatgactc
agattttaaa ttagatgctt taaaagaaaa aacaattgca 60gtaatcggtt atggaagtca
aggtagggca cagtccttaa acatgaaaga cagcggatta 120aacgttgttg
ttggtttaag aaaaaacggt gcttcatggg aaaacgctaa agcagacggt
180cacaacgtaa tgactatcga agaagctgct gaaaaagctg acatcatcca
catcttaatt 240cctgacgaat tacaggcaga agtttatgaa agccagataa
aaccatattt aaaggaagga 300aaaacactca gcttttcaca tggttttaac
atccactatg gattcattgt tccaccaaag 360ggagttaacg tggttttagt
tgctccaaaa tcacctggaa aaatggttag aagaacatac 420gaagaaggct
tcggtgttcc aggtttaatc tgtatcgaaa tcgatgcaac aaacaacgca
480tttgacattg tttcagcaat ggcaaaagga atcggtttat caagggccgg
agttatccag 540acaactttca aagaagaaac agaaactgac cttttcggtg
aacaagctgt tttatgcggc 600ggagttaccg aattaatcaa agcaggattc
gaaacacttg ttgaagcagg atacgcacca 660gaaatggcat actttgaaac
atgccacgaa ttgaaattaa ttgttgactt aatctaccaa 720aaaggattca
aaaacatgtg gaacgatgta agtaacactg cagaatacgg tggacttaca
780agaagaagca gaatcgttac agctgactca aaagctgcaa tgaaagaaat
cttaaaagaa 840atccaagatg gaagattcac aaaagaattc gtgcttgaaa
aacaagtaaa ccacgcgcac 900ttaaaagcaa tgagaagaat cgaaggagac
ttacaaatcg aagaagtcgg tgcaaaatta 960agaaaaatgt gcggtcttga
aaaagaagaa tga 99328330PRTMethanococcus maripaludis 28Met Lys Val
Phe Tyr Asp Ser Asp Phe Lys Leu Asp Ala Leu Lys Glu 1 5 10 15 Lys
Thr Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly Arg Ala Gln Ser 20 25
30 Leu Asn Met Lys Asp Ser Gly Leu Asn Val Val Val Gly Leu Arg Lys
35 40 45 Asn Gly Ala Ser Trp Glu Asn Ala Lys Ala Asp Gly His Asn
Val Met 50 55 60 Thr Ile Glu Glu Ala Ala Glu Lys Ala Asp Ile Ile
His Ile Leu Ile 65 70 75 80 Pro Asp Glu Leu Gln Ala Glu Val Tyr Glu
Ser Gln Ile Lys Pro Tyr 85 90 95 Leu Lys Glu Gly Lys Thr Leu Ser
Phe Ser His Gly Phe Asn Ile His 100 105 110 Tyr Gly Phe Ile Val Pro
Pro Lys Gly Val Asn Val Val Leu Val Ala 115 120 125 Pro Lys Ser Pro
Gly Lys Met Val Arg Arg Thr Tyr Glu Glu Gly Phe 130 135 140 Gly Val
Pro Gly Leu Ile Cys Ile Glu Ile Asp Ala Thr Asn Asn Ala 145 150 155
160 Phe Asp Ile Val Ser Ala Met Ala Lys Gly Ile Gly Leu Ser Arg Ala
165 170 175 Gly Val Ile Gln Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp
Leu Phe 180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Val Thr Glu
Leu Ile Lys Ala 195 200 205 Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr
Ala Pro Glu Met Ala Tyr 210 215 220 Phe Glu Thr Cys His Glu Leu Lys
Leu Ile Val Asp Leu Ile Tyr Gln 225 230 235 240 Lys Gly Phe Lys Asn
Met Trp Asn Asp Val Ser Asn Thr Ala Glu Tyr 245 250 255 Gly Gly Leu
Thr Arg Arg Ser Arg Ile Val Thr Ala Asp Ser Lys Ala 260 265 270 Ala
Met Lys Glu Ile Leu Lys Glu Ile Gln Asp Gly Arg Phe Thr Lys 275 280
285 Glu Phe Val Leu Glu Lys Gln Val Asn His Ala His Leu Lys Ala Met
290 295 300 Arg Arg Ile Glu Gly Asp Leu Gln Ile Glu Glu Val Gly Ala
Lys Leu 305 310 315 320 Arg Lys Met Cys Gly Leu Glu Lys Glu Glu 325
330 29993DNAMethanococcus maripaludis 29atgaaggtat tctatgactc
agattttaaa ttagatgctt taaaagaaaa aacaattgca 60gtaatcggtt acggaagtca
aggaagagca cagtccttaa acatgaaaga cagtggatta 120aacgttgttg
ttggtttaag gaaaaatgga gcttcgtggg aaaacgctaa agcagacggt
180cacaatgtaa tgactatcga agaagctgct gaaaaagctg acatcatcca
catcttaatc 240cctgacgaat tacaggcaga agtttacgat gctcaaataa
aaccatacct caaagaagga 300aaaacactca gtttctcaca tggttttaac
atccactatg gattcattgt tccaccaaaa 360ggagttaacg tggttttagt
tgctccaaaa tcacctggaa aaatggttag aagaacatac 420gaagaaggtt
tcggtgttcc aggtttaatc tgtatcgaaa tagatgcaac aaacaacgca
480tttgacattg tttcagcaat ggcaaaagga atcggtttat caagagctgg
agttatccag 540acaacattta aagaagaaac agaaactgac cttttcggtg
aacaagctgt tttatgcggt 600ggagttaccg aattaatcaa agcaggattt
gaaacactcg tagaagcagg atacgcacca 660gaaatggcat actttgaaac
atgccacgaa ttgaaattaa tcgttgactt aatctaccaa 720aaaggattca
aaaacatgtg gaacgacgta agtaacactg cagaatacgg tggacttaca
780agaagaagca gaattgtaac tgctgattca aaagctgcaa tgaaagaaat
cttaaaagaa 840atccaagacg gaagattcac aaaagaattc gttcttgaaa
aacaagtaaa ccacgcacat 900ttaaaagcaa tgagaagact cgaaggagaa
ttacaaatcg aagaagtcgg tgcaaaatta 960agaaaaatgt gcggtcttga
aaaagaagaa taa 99330330PRTMethanococcus maripaludis 30Met Lys Val
Phe Tyr Asp Ser Asp Phe Lys Leu Asp Ala Leu Lys Glu 1 5 10 15 Lys
Thr Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly Arg Ala Gln Ser 20 25
30 Leu Asn Met Lys Asp Ser Gly Leu Asn Val Val Val Gly Leu Arg Lys
35 40 45 Asn Gly Ala Ser Trp Glu Asn Ala Lys Ala Asp Gly His Asn
Val Met 50 55 60 Thr Ile Glu Glu Ala Ala Glu Lys Ala Asp Ile Ile
His Ile Leu Ile 65 70 75 80 Pro Asp Glu Leu Gln Ala Glu Val Tyr Asp
Ala Gln Ile Lys Pro Tyr 85 90 95 Leu Lys Glu Gly Lys Thr Leu Ser
Phe Ser His Gly Phe Asn Ile His 100 105 110 Tyr Gly Phe Ile Val Pro
Pro Lys Gly Val Asn Val Val Leu Val Ala 115 120 125 Pro Lys Ser Pro
Gly Lys Met Val Arg Arg Thr Tyr Glu Glu Gly Phe 130 135 140 Gly Val
Pro Gly Leu Ile Cys Ile Glu Ile Asp Ala Thr Asn Asn Ala 145 150 155
160 Phe Asp Ile Val Ser Ala Met Ala Lys Gly Ile Gly Leu Ser Arg Ala
165 170 175 Gly Val Ile Gln Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp
Leu Phe 180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Val Thr Glu
Leu Ile Lys Ala 195 200 205 Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr
Ala Pro Glu Met Ala Tyr 210 215 220 Phe Glu Thr Cys His Glu Leu Lys
Leu Ile Val Asp Leu Ile Tyr Gln 225 230 235 240 Lys Gly Phe Lys Asn
Met Trp Asn Asp Val Ser Asn Thr Ala Glu Tyr 245 250 255 Gly Gly Leu
Thr Arg Arg Ser Arg Ile Val Thr Ala Asp Ser Lys Ala 260 265 270 Ala
Met Lys Glu Ile Leu Lys Glu Ile Gln Asp Gly Arg Phe Thr Lys 275 280
285 Glu Phe Val Leu Glu Lys Gln Val Asn His Ala His Leu Lys Ala Met
290 295 300 Arg Arg Leu Glu Gly Glu Leu Gln Ile Glu Glu Val Gly Ala
Lys Leu 305 310 315 320 Arg Lys Met Cys Gly Leu Glu Lys Glu Glu 325
330 31993DNAMethanococcus maripaludis 31atgaaggtat tctatgactc
agattttaaa ttagatgctt taaaagaaaa aacaatcgca 60gtaatcggtt acggaagcca
aggtcgggca cagtccttaa acatgaaaga cagcggatta 120aacgttgttg
ttggtttaag aaaaaatggt gcttcatggg aaaacgctaa agcagacggt
180cacaatgtaa tgaccatcga agaagctgct gaaaaagctg acatcatcca
catcttaatc 240cctgacgaat tacaggcaga agtttacgat gctcaaataa
aaccatgcct caaagaagga 300aaaacactca gcttttcaca tggttttaac
atccactatg gattcattgt tccaccaaaa 360ggagttaacg tggttttagt
tgctccaaaa tcaccaggaa aaatggttag aagaacatac 420gaagaaggtt
tcggtgttcc aggattaatc tgtatcgaaa ttgatgcaac aaacaatgca
480tttgacattg tttcagcaat ggcaaaagga atcggtttat caagagctgg
agttatccag 540acaacattta aagaagaaac agaaaccgat cttttcggtg
aacaagctgt tttatgcggt 600ggagttaccg aattaatcaa agcaggattt
gaaacactcg ttgaagcagg atacgcacca 660gaaatggctt actttgaaac
atgtcacgaa ttgaaattaa ttgttgactt aatctaccaa 720aaaggattca
aaaacatgtg gaacgatgta agtaacactg ctgaatatgg tggacttaca
780agaagaagca gaattgttac tgctgactca aaagctgcaa tgaaagaaat
tttaaaagaa 840atccaagacg gaagattcac aaaagaattc gtgcttgaaa
aacaagtaaa ccacgcgcac 900ttaaaagcaa tgagaagaat cgaaggagaa
ttacaaatcg aggaagtcgg cgcaaaatta 960agaaaaatgt gcggtcttga
aaaagaagaa taa 99332330PRTMethanococcus maripaludis 32Met Lys Val
Phe Tyr Asp Ser Asp Phe Lys Leu Asp Ala Leu Lys Glu 1 5 10 15 Lys
Thr Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly Arg Ala Gln Ser 20 25
30 Leu Asn Met Lys Asp Ser Gly Leu Asn Val Val Val Gly Leu Arg Lys
35 40 45 Asn Gly Ala Ser Trp Glu Asn Ala Lys Ala Asp Gly His Asn
Val Met 50 55 60 Thr Ile Glu Glu Ala Ala Glu Lys Ala Asp Ile Ile
His Ile Leu Ile 65 70 75 80 Pro Asp Glu Leu Gln Ala Glu Val Tyr Asp
Ala Gln Ile Lys Pro Cys 85 90 95 Leu Lys Glu Gly Lys Thr Leu Ser
Phe Ser His Gly Phe Asn Ile His 100 105 110 Tyr Gly Phe Ile Val Pro
Pro Lys Gly Val Asn Val Val Leu Val Ala 115 120 125 Pro Lys Ser Pro
Gly Lys Met Val Arg Arg Thr Tyr Glu Glu Gly Phe 130 135 140 Gly Val
Pro Gly Leu Ile Cys Ile Glu Ile Asp Ala Thr Asn Asn Ala 145 150 155
160 Phe Asp Ile Val Ser Ala Met Ala Lys Gly Ile Gly Leu Ser Arg Ala
165 170 175 Gly Val Ile Gln Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp
Leu Phe 180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Val Thr Glu
Leu Ile Lys Ala 195 200 205 Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr
Ala Pro Glu Met Ala Tyr 210 215 220 Phe Glu Thr Cys His Glu Leu Lys
Leu Ile Val Asp Leu Ile Tyr Gln 225 230 235 240 Lys Gly Phe Lys Asn
Met Trp Asn Asp Val Ser Asn Thr Ala Glu Tyr 245 250 255 Gly Gly Leu
Thr Arg Arg Ser Arg Ile Val Thr Ala Asp Ser Lys Ala 260 265 270 Ala
Met Lys Glu Ile Leu Lys Glu Ile Gln Asp Gly Arg Phe Thr Lys 275 280
285 Glu Phe Val Leu Glu Lys Gln Val Asn His Ala His Leu Lys Ala Met
290 295 300 Arg Arg Ile Glu Gly Glu Leu Gln Ile Glu Glu Val Gly Ala
Lys Leu 305 310 315 320 Arg Lys Met Cys Gly Leu Glu Lys Glu Glu 325
330 33993DNAMethanococcus maripaludis 33atgaaggtat tctatgactc
agattttaaa ttagatgctt taaaagaaaa aacaattgca 60gtaatcggtt atggaagtca
aggtagggca cagtccttaa acatgaaaga cagcggatta 120aacgttgttg
ttggtttaag aaaaaacggt gcttcatgga acaacgctaa agcagacggt
180cacaatgtaa tgaccattga agaagctgct gaaaaagcgg acatcatcca
catcttaata 240cctgatgaat tacaggcaga agtttatgaa agccagataa
aaccatacct aaaagaagga 300aaaacactaa gcttttcaca tggttttaac
atccactatg gattcattgt tccaccaaaa 360ggagttaacg tggttttagt
tgctccaaaa tcacctggaa aaatggttag aagaacatac 420gaagaaggtt
tcggtgttcc aggtttaatc tgtattgaaa ttgatgcaac aaacaacgca
480tttgatattg tttcagcaat ggcaaaagga atcggtttat caagagctgg
agttatccag 540acaactttca aagaagaaac agaaactgac cttttcggtg
aacaagctgt tttatgcggt 600ggagttaccg aattaatcaa ggcaggattt
gaaacactcg ttgaagcagg atacgcacca 660gaaatggcat actttgaaac
ctgccacgaa ttgaaattaa tcgttgactt aatctaccaa 720aaaggattca
aaaacatgtg gaacgatgta agtaacactg cagaatacgg cggacttaca
780agaagaagca gaatcgttac agctgattca aaagctgcaa tgaaagaaat
cttaagagaa 840atccaagatg gaagattcac aaaagaattc cttctcgaaa
aacaggtaag ctatgctcat 900ttaaaatcaa tgagaagact cgaaggagac
ttacaaatcg aagaagtcgg cgcaaaatta 960agaaaaatgt gcggtcttga
aaaagaagaa taa 99334330PRTMethanococcus maripaludis 34Met Lys Val
Phe Tyr Asp Ser Asp Phe Lys Leu Asp Ala Leu Lys Glu 1 5 10 15 Lys
Thr Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly Arg Ala Gln Ser 20
25 30 Leu Asn Met Lys Asp Ser Gly Leu Asn Val Val Val Gly Leu Arg
Lys 35 40 45 Asn Gly Ala Ser Trp Asn Asn Ala Lys Ala Asp Gly His
Asn Val Met 50 55 60 Thr Ile Glu Glu Ala Ala Glu Lys Ala Asp Ile
Ile His Ile Leu Ile 65 70 75 80 Pro Asp Glu Leu Gln Ala Glu Val Tyr
Glu Ser Gln Ile Lys Pro Tyr 85 90 95 Leu Lys Glu Gly Lys Thr Leu
Ser Phe Ser His Gly Phe Asn Ile His 100 105 110 Tyr Gly Phe Ile Val
Pro Pro Lys Gly Val Asn Val Val Leu Val Ala 115 120 125 Pro Lys Ser
Pro Gly Lys Met Val Arg Arg Thr Tyr Glu Glu Gly Phe 130 135 140 Gly
Val Pro Gly Leu Ile Cys Ile Glu Ile Asp Ala Thr Asn Asn Ala 145 150
155 160 Phe Asp Ile Val Ser Ala Met Ala Lys Gly Ile Gly Leu Ser Arg
Ala 165 170 175 Gly Val Ile Gln Thr Thr Phe Lys Glu Glu Thr Glu Thr
Asp Leu Phe 180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Val Thr
Glu Leu Ile Lys Ala 195 200 205 Gly Phe Glu Thr Leu Val Glu Ala Gly
Tyr Ala Pro Glu Met Ala Tyr 210 215 220 Phe Glu Thr Cys His Glu Leu
Lys Leu Ile Val Asp Leu Ile Tyr Gln 225 230 235 240 Lys Gly Phe Lys
Asn Met Trp Asn Asp Val Ser Asn Thr Ala Glu Tyr 245 250 255 Gly Gly
Leu Thr Arg Arg Ser Arg Ile Val Thr Ala Asp Ser Lys Ala 260 265 270
Ala Met Lys Glu Ile Leu Arg Glu Ile Gln Asp Gly Arg Phe Thr Lys 275
280 285 Glu Phe Leu Leu Glu Lys Gln Val Ser Tyr Ala His Leu Lys Ser
Met 290 295 300 Arg Arg Leu Glu Gly Asp Leu Gln Ile Glu Glu Val Gly
Ala Lys Leu 305 310 315 320 Arg Lys Met Cys Gly Leu Glu Lys Glu Glu
325 330 35993DNAMethanococcus vannielii 35atgaaggtat tctacgatgc
agacataaaa ttagacgctt taaaaagtaa aacaattgca 60gttattggtt acggaagtca
gggtagagcc cagtctttaa acatgaaaga cagcggttta 120aacgttgtag
ttggtttaag gaaaaacggt gcttcatggg aaaacgctaa aaacgatggt
180catgaagtat taacgattga agaagcttca aaaaaagcag acataattca
tatattaatc 240cctgatgaat tacaggctga agtttacgaa agccagataa
aaccatacct tacagaagga 300aaaacattaa gcttctcaca cggctttaat
atccattatg ggtttattat tccgccaaaa 360ggagttaacg tggttttagt
tgcaccaaag tcacccggta aaatggttag aaaaacatac 420gaagaaggat
ttggtgttcc gggattaatc tgtatagaag tagatgctac aaatactgca
480tttgagactg tttcagcaat ggcaaagggg atcggcctct caagagcagg
cgttatccag 540acaacattta gggaggaaac tgaaaccgat ctttttggtg
aacaggcagt attgtgcggc 600ggagttactg aattaattaa agcaggattt
gaaacactcg ttgaagcagg atattcacct 660gaaatggcgt attttgaaac
atgccacgag ttaaaattaa ttgttgactt aatttaccaa 720aaaggattca
aaaacatgtg gcatgatgta agtaatactg cagaatatgg tggacttaca
780agaagaagca gaatcgttac tgctgactca aaagctgcga tgaaagaaat
tttaaaagag 840attcaagatg gaagatttac aaaagaattt gttcttgaaa
atcaagctaa aatggcacat 900ttaaaagcaa tgaggagact tgaaggcgaa
ttgcaaattg aagaagtcgg ttcaaagtta 960agaaaaatgt gtggtcttga
aaaagacgaa taa 99336330PRTMethanococcus vannielii 36Met Lys Val Phe
Tyr Asp Ala Asp Ile Lys Leu Asp Ala Leu Lys Ser 1 5 10 15 Lys Thr
Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly Arg Ala Gln Ser 20 25 30
Leu Asn Met Lys Asp Ser Gly Leu Asn Val Val Val Gly Leu Arg Lys 35
40 45 Asn Gly Ala Ser Trp Glu Asn Ala Lys Asn Asp Gly His Glu Val
Leu 50 55 60 Thr Ile Glu Glu Ala Ser Lys Lys Ala Asp Ile Ile His
Ile Leu Ile 65 70 75 80 Pro Asp Glu Leu Gln Ala Glu Val Tyr Glu Ser
Gln Ile Lys Pro Tyr 85 90 95 Leu Thr Glu Gly Lys Thr Leu Ser Phe
Ser His Gly Phe Asn Ile His 100 105 110 Tyr Gly Phe Ile Ile Pro Pro
Lys Gly Val Asn Val Val Leu Val Ala 115 120 125 Pro Lys Ser Pro Gly
Lys Met Val Arg Lys Thr Tyr Glu Glu Gly Phe 130 135 140 Gly Val Pro
Gly Leu Ile Cys Ile Glu Val Asp Ala Thr Asn Thr Ala 145 150 155 160
Phe Glu Thr Val Ser Ala Met Ala Lys Gly Ile Gly Leu Ser Arg Ala 165
170 175 Gly Val Ile Gln Thr Thr Phe Arg Glu Glu Thr Glu Thr Asp Leu
Phe 180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Val Thr Glu Leu
Ile Lys Ala 195 200 205 Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr Ser
Pro Glu Met Ala Tyr 210 215 220 Phe Glu Thr Cys His Glu Leu Lys Leu
Ile Val Asp Leu Ile Tyr Gln 225 230 235 240 Lys Gly Phe Lys Asn Met
Trp His Asp Val Ser Asn Thr Ala Glu Tyr 245 250 255 Gly Gly Leu Thr
Arg Arg Ser Arg Ile Val Thr Ala Asp Ser Lys Ala 260 265 270 Ala Met
Lys Glu Ile Leu Lys Glu Ile Gln Asp Gly Arg Phe Thr Lys 275 280 285
Glu Phe Val Leu Glu Asn Gln Ala Lys Met Ala His Leu Lys Ala Met 290
295 300 Arg Arg Leu Glu Gly Glu Leu Gln Ile Glu Glu Val Gly Ser Lys
Leu 305 310 315 320 Arg Lys Met Cys Gly Leu Glu Lys Asp Glu 325 330
37990DNAMethanococcus voltae 37atgcaagtac tttacgaagc tgacgcaaac
tatgataaat taaaaggcaa aacaatagca 60gttatcggat acggtagcca aggtagagct
caatcattaa atatgaaaga gagcggttta 120aacgtaataa tgggtttaag
agaaggcggt gcatcctggg aatctgctaa aaaagacggc 180cacgaagtat
actcaatcga ggaagctgca aaaatggctg acgttataca tatattaata
240cctgacgaaa tccaaggtaa tgtatacaat agccaaataa aaccttattt
ggaagaaggc 300aacacattaa gcttttcaca cggttataat atccatttta
actacattgt agcaccaaaa 360ggtgttaata taacaatggt agctcctaaa
tcacctggaa aaatggtaag aaaaacctac 420gaagaaggtt tcggtgtacc
tggtttaatc tgcatcgaaa aagacgaaac tggcgaagct 480tacgatattg
cattaggtat ggcaaaaggt atcggattaa caagagcggg agttattcaa
540acaacattca gggaagaaac agaaaccgat ttattcggtg agcaagctgt
tctctgtggt 600ggcgttactg aattaatcaa agcaggattt gagacacttg
ttgaagcagg atatgctcca 660gaaatggctt actttgaaac ttgccacgaa
ttgaaattaa tcgttgattt aatctaccaa 720aaaggattta aaaatatgtg
gcacgatgta agtaatactg cggaatatgg tggacttacc 780agaagagaaa
gagtagttac aaaagaatca aaagaagcaa tgaaggaaat cttaaaagaa
840atccaagatg gaagatttac aaaagaattt gctctcgaaa accaagctgg
aaaacctcac 900ttaaattcaa tgagaagatt agaaggagaa ttactcatcg
aacaagtagg tgctgattta 960aggaaaaaat gcggtttaga aaaagaataa
99038329PRTMethanococcus voltae 38Met Gln Val Leu Tyr Glu Ala Asp
Ala Asn Tyr Asp Lys Leu Lys Gly 1 5 10 15 Lys Thr Ile Ala Val Ile
Gly Tyr Gly Ser Gln Gly Arg Ala Gln Ser 20 25 30 Leu Asn Met Lys
Glu Ser Gly Leu Asn Val Ile Met Gly Leu Arg Glu 35 40 45 Gly Gly
Ala Ser Trp Glu Ser Ala Lys Lys Asp Gly His Glu Val Tyr 50 55 60
Ser Ile Glu Glu Ala Ala Lys Met Ala Asp Val Ile His Ile Leu Ile 65
70 75 80 Pro Asp Glu Ile Gln Gly Asn Val Tyr Asn Ser Gln Ile Lys
Pro Tyr 85 90 95 Leu Glu Glu Gly Asn Thr Leu Ser Phe Ser His Gly
Tyr Asn Ile His 100 105 110 Phe Asn Tyr Ile Val Ala Pro Lys Gly Val
Asn Ile Thr Met Val Ala 115 120 125 Pro Lys Ser Pro Gly Lys Met Val
Arg Lys Thr Tyr Glu Glu Gly Phe 130 135 140 Gly Val Pro Gly Leu Ile
Cys Ile Glu Lys Asp Glu Thr Gly Glu Ala 145 150 155 160 Tyr Asp Ile
Ala Leu Gly Met Ala Lys Gly Ile Gly Leu Thr Arg Ala 165 170 175 Gly
Val Ile Gln Thr Thr Phe Arg Glu Glu Thr Glu Thr Asp Leu Phe 180 185
190 Gly Glu Gln Ala Val Leu Cys Gly Gly Val Thr Glu Leu Ile Lys Ala
195 200 205 Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr Ala Pro Glu Met
Ala Tyr 210 215 220 Phe Glu Thr Cys His Glu Leu Lys Leu Ile Val Asp
Leu Ile Tyr Gln 225 230 235 240 Lys Gly Phe Lys Asn Met Trp His Asp
Val Ser Asn Thr Ala Glu Tyr 245 250 255 Gly Gly Leu Thr Arg Arg Glu
Arg Val Val Thr Lys Glu Ser Lys Glu 260 265 270 Ala Met Lys Glu Ile
Leu Lys Glu Ile Gln Asp Gly Arg Phe Thr Lys 275 280 285 Glu Phe Ala
Leu Glu Asn Gln Ala Gly Lys Pro His Leu Asn Ser Met 290 295 300 Arg
Arg Leu Glu Gly Glu Leu Leu Ile Glu Gln Val Gly Ala Asp Leu 305 310
315 320 Arg Lys Lys Cys Gly Leu Glu Lys Glu 325 391020DNAZymomonas
mobilis 39atgaaagttt attacgatag tgatgctgat cttgggctga tcaagtccaa
gaaaatcgct 60attcttggct atggtagcca gggtcacgcc catgcacaga atttgcgcga
ttccggtgtt 120gctgaagtag ctattgcgct tcgtcctgat tcggcttctg
ttaaaaaagc acaggatgct 180ggtttcaagg ttttgaccaa tgctgaagcc
gcaaaatggg ctgatatcct gatgatcttg 240gcacctgatg aacatcaggc
tgctatctat gccgaagatt taaaagataa tttgcgccct 300ggtagtgcaa
ttgcttttgc tcatggtttg aatatccatt tcggtctgat cgaaccccgc
360aaagatatcg atgttttcat gatcgcaccg aaaggcccag gtcacacggt
tcgttctgaa 420tatgtccgtg gcggtggtgt gccttgcttg gtcgccgttg
atcaggatgc cagcggtaac 480gctcatgaca tcgctcttgc ttatgcttct
ggcatcggtg gcggtcgttc tggtgttatt 540gaaaccactt tccgtgaaga
agtcgaaacc gatttgtttg gtgagcaggc tgttctctgc 600ggtggtttga
ctgcgcttat cacggctggt tttgaaactt tgactgaagc cggttacgct
660cctgaaatgg cattcttcga atgtatgcat gaaatgaagc tgatcgtgga
tctgatctac 720gaagcgggta ttgccaatat gcgttattcg atttctaaca
ctgccgaata tggtgatatc 780gtatctggcc cgcgggtcat caatgaagaa
tccaaaaagg caatgaaggc tattctggac 840gacatccaga gcggtcgttt
tgtcagcaaa tttgttcttg ataaccgcgc tggtcagccg 900gaactcaaag
ctgcccgtaa acgtatggct gctcacccga tcgaacaggt tggtgcacgt
960ctgcgtaaaa tgatgccgtg gatcgccagc aacaagctgg ttgataaggc
tcgcaactag 102040339PRTZymomonas mobilis 40Met Lys Val Tyr Tyr Asp
Ser Asp Ala Asp Leu Gly Leu Ile Lys Ser 1 5 10 15 Lys Lys Ile Ala
Ile Leu Gly Tyr Gly Ser Gln Gly His Ala His Ala 20 25 30 Gln Asn
Leu Arg Asp Ser Gly Val Ala Glu Val Ala Ile Ala Leu Arg 35 40 45
Pro Asp Ser Ala Ser Val Lys Lys Ala Gln Asp Ala Gly Phe Lys Val 50
55 60 Leu Thr Asn Ala Glu Ala Ala Lys Trp Ala Asp Ile Leu Met Ile
Leu 65 70 75 80 Ala Pro Asp Glu His Gln Ala Ala Ile Tyr Ala Glu Asp
Leu Lys Asp 85 90 95 Asn Leu Arg Pro Gly Ser Ala Ile Ala Phe Ala
His Gly Leu Asn Ile 100 105 110 His Phe Gly Leu Ile Glu Pro Arg Lys
Asp Ile Asp Val Phe Met Ile 115 120 125 Ala Pro Lys Gly Pro Gly His
Thr Val Arg Ser Glu Tyr Val Arg Gly 130 135 140 Gly Gly Val Pro Cys
Leu Val Ala Val Asp Gln Asp Ala Ser Gly Asn 145 150 155 160 Ala His
Asp Ile Ala Leu Ala Tyr Ala Ser Gly Ile Gly Gly Gly Arg 165 170 175
Ser Gly Val Ile Glu Thr Thr Phe Arg Glu Glu Val Glu Thr Asp Leu 180
185 190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Thr Ala Leu Ile
Thr 195 200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Ala Pro
Glu Met Ala 210 215 220 Phe Phe Glu Cys Met His Glu Met Lys Leu Ile
Val Asp Leu Ile Tyr 225 230 235 240 Glu Ala Gly Ile Ala Asn Met Arg
Tyr Ser Ile Ser Asn Thr Ala Glu 245 250 255 Tyr Gly Asp Ile Val Ser
Gly Pro Arg Val Ile Asn Glu Glu Ser Lys 260 265 270 Lys Ala Met Lys
Ala Ile Leu Asp Asp Ile Gln Ser Gly Arg Phe Val 275 280 285 Ser Lys
Phe Val Leu Asp Asn Arg Ala Gly Gln Pro Glu Leu Lys Ala 290 295 300
Ala Arg Lys Arg Met Ala Ala His Pro Ile Glu Gln Val Gly Ala Arg 305
310 315 320 Leu Arg Lys Met Met Pro Trp Ile Ala Ser Asn Lys Leu Val
Asp Lys 325 330 335 Ala Arg Asn 411020DNAErythrobacter sp.
41atgaaagtct actacgacgc cgatgccgat cttggcctca tcaaatccaa gaagatcgcc
60gtgctcggct atggctcgca gggccacgct cacgcccaga acctgcgcga cagcggtgtc
120gccgaagtgg caattgccct tcgcgaaggc tcggccacag caaagaaggc
gcaagatgca 180ggcttcaagg tgctttccaa taccgaggct gccaagtggg
ccgatatcgt gatgatcctc 240gcacccgatg agcatcaggc agcaatctgg
gaaaatgatc tcgccggcca catgaagccg 300ggcagcgcga ttgcctttgc
ccacggcctc aacattcact tcggccttat cgaagcaccg 360caggatatcg
acgtcatcat gatcgcgccc aaaggtccgg ggcacactgt gcgtagcgaa
420taccagcgcg gcggcggcgt cccttgcctg attgctgttc atcaggacgc
gagcggcagc 480gccaaggaaa tcgccctcgc ctacgcatca ggcgtcggag
gcggccgctc gggcatcatc 540gagaccaact tccgcgagga atgcgagacc
gatctgttcg gtgagcaggc cgtgctttgc 600ggcgggatca cgcacctgat
tcaggccggt ttcgaaaccc tgaccgaagc cggttacgcg 660cccgaaatgg
cctatttcga gtgcctccac gaaaccaagc tgatcgtcga tcttctctac
720gaaggcggca ttgcgaacat gcgctattcc atctcgaaca cggcggaata
tggcgacatc 780accaccggcc cgcgcatcat caccgatgag acgaaggccg
agatgaagcg cgtgctcgac 840gacatccaat cgggccgctt cgtgaagaac
ttcgtgctcg acaatcgcgc aggccagccc 900gaactcaagg cagcccgcaa
gcgcgccgaa gctcacccga ttgagaagac cggcgcagaa 960ctgcgcgcaa
tgatgccatg gatcagtgcc aacaagctgg tcgacaagtc gaaaaactag
102042339PRTErythrobacter sp. 42Met Lys Val Tyr Tyr Asp Ala Asp Ala
Asp Leu Gly Leu Ile Lys Ser 1 5 10 15 Lys Lys Ile Ala Val Leu Gly
Tyr Gly Ser Gln Gly His Ala His Ala 20 25 30 Gln Asn Leu Arg Asp
Ser Gly Val Ala Glu Val Ala Ile Ala Leu Arg 35 40 45 Glu Gly Ser
Ala Thr Ala Lys Lys Ala Gln Asp Ala Gly Phe Lys Val 50 55 60 Leu
Ser Asn Thr Glu Ala Ala Lys Trp Ala Asp Ile Val Met Ile Leu 65 70
75 80 Ala Pro Asp Glu His Gln Ala Ala Ile Trp Glu Asn Asp Leu Ala
Gly 85 90 95 His Met Lys Pro Gly Ser Ala Ile Ala Phe Ala His Gly
Leu Asn Ile 100 105 110 His Phe Gly Leu Ile Glu Ala Pro Gln Asp Ile
Asp Val Ile Met Ile 115 120 125 Ala Pro Lys Gly Pro Gly His Thr Val
Arg Ser Glu Tyr Gln Arg Gly 130 135 140 Gly Gly Val Pro Cys Leu Ile
Ala Val His Gln Asp Ala Ser Gly Ser 145 150 155 160 Ala Lys Glu Ile
Ala Leu Ala Tyr Ala Ser Gly Val Gly Gly Gly Arg 165 170 175 Ser Gly
Ile Ile Glu Thr Asn Phe Arg Glu Glu Cys Glu Thr Asp Leu 180 185 190
Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Ile Thr His Leu Ile Gln 195
200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Ala Pro Glu Met
Ala 210 215 220 Tyr Phe Glu Cys Leu His Glu Thr Lys Leu Ile Val Asp
Leu Leu Tyr 225 230 235 240 Glu Gly Gly Ile Ala Asn Met Arg Tyr Ser
Ile Ser Asn Thr Ala Glu 245 250 255 Tyr Gly Asp Ile Thr Thr Gly Pro
Arg Ile Ile Thr Asp Glu Thr Lys 260 265 270 Ala Glu Met Lys Arg Val
Leu Asp Asp Ile Gln Ser Gly Arg Phe Val 275 280 285 Lys Asn Phe Val
Leu Asp Asn Arg Ala Gly Gln Pro Glu Leu Lys Ala 290 295 300 Ala Arg
Lys Arg Ala Glu Ala His Pro Ile Glu Lys Thr Gly Ala Glu 305 310 315
320 Leu Arg Ala Met Met Pro Trp Ile Ser Ala Asn Lys Leu Val Asp Lys
325 330 335 Ser Lys Asn 431020DNASphingomonas wittichii
43atgcgcgtct
attatgatcg tgatgccgac atcggcctca tcaagaccaa gaaggtggcg 60atcgtcggct
atggcagcca gggccacgcc catgcccaga acctgcagga ctcgggcgtc
120gccgacgtcg cgatcgcgct gcgccccggc tcggccaccg cgaagaaggc
cgaaggcgcc 180ggcttcaagg tgctgtcgaa cgccgacgcg gccaagtggg
ccgacatcgt catgatcctg 240gcgcccgacg agcaccaggc cgcgatctac
aatgacgacc tgcgcgacaa tctgaagccg 300ggcgcggcgc tcgccttcgc
ccatggcctc aacgtccatt tcggcctgat cgagccgcgc 360gccgacatcg
acgtgttcat gatcgcgccg aagggccccg gccacaccgt ccgttccgaa
420tatcagcgcg gcggcggcgt gccctgcctg atcgcgatcg cccaggacgc
cagcggcaac 480gcgcacgacg tcgccctgtc ctacgcctcg gcgatcggcg
gcggccgttc gggcgtgatc 540gagacgacct tcaaggaaga gtgcgagacc
gacctgttcg gcgagcaggc ggtgctgtgc 600ggcggcctca gccacctgat
catggccggc ttcgagacgc tggtcgaggc gggctacgcc 660cccgagatgg
cctatttcga atgcctccac gaagtgaagc tgatcgtcga cctgatgtat
720gagggcggca tcgccaacat gcgctactcg atctcgaaca ccgccgaata
tggcgacatc 780cacaccggcc cgcgcgtcat cacctcggag accaaggccg
agatgaagcg cgtgctcgac 840gacatccaga agggcaagtt cgtcaagcgc
ttcgtcctcg acaaccgcgc cggccagccc 900gagctgaagg cgagccgcaa
gctcgtcgcc gagcatccga tcgagaaggt cggcgccgaa 960ctgcgcgcga
tgatgccctg gatcagcaag aaccagctgg tcgacaaggc caagaactga
102044339PRTSphingomonas wittichii 44Met Arg Val Tyr Tyr Asp Arg
Asp Ala Asp Ile Gly Leu Ile Lys Thr 1 5 10 15 Lys Lys Val Ala Ile
Val Gly Tyr Gly Ser Gln Gly His Ala His Ala 20 25 30 Gln Asn Leu
Gln Asp Ser Gly Val Ala Asp Val Ala Ile Ala Leu Arg 35 40 45 Pro
Gly Ser Ala Thr Ala Lys Lys Ala Glu Gly Ala Gly Phe Lys Val 50 55
60 Leu Ser Asn Ala Asp Ala Ala Lys Trp Ala Asp Ile Val Met Ile Leu
65 70 75 80 Ala Pro Asp Glu His Gln Ala Ala Ile Tyr Asn Asp Asp Leu
Arg Asp 85 90 95 Asn Leu Lys Pro Gly Ala Ala Leu Ala Phe Ala His
Gly Leu Asn Val 100 105 110 His Phe Gly Leu Ile Glu Pro Arg Ala Asp
Ile Asp Val Phe Met Ile 115 120 125 Ala Pro Lys Gly Pro Gly His Thr
Val Arg Ser Glu Tyr Gln Arg Gly 130 135 140 Gly Gly Val Pro Cys Leu
Ile Ala Ile Ala Gln Asp Ala Ser Gly Asn 145 150 155 160 Ala His Asp
Val Ala Leu Ser Tyr Ala Ser Ala Ile Gly Gly Gly Arg 165 170 175 Ser
Gly Val Ile Glu Thr Thr Phe Lys Glu Glu Cys Glu Thr Asp Leu 180 185
190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Ser His Leu Ile Met
195 200 205 Ala Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr Ala Pro Glu
Met Ala 210 215 220 Tyr Phe Glu Cys Leu His Glu Val Lys Leu Ile Val
Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Ile Ala Asn Met Arg Tyr
Ser Ile Ser Asn Thr Ala Glu 245 250 255 Tyr Gly Asp Ile His Thr Gly
Pro Arg Val Ile Thr Ser Glu Thr Lys 260 265 270 Ala Glu Met Lys Arg
Val Leu Asp Asp Ile Gln Lys Gly Lys Phe Val 275 280 285 Lys Arg Phe
Val Leu Asp Asn Arg Ala Gly Gln Pro Glu Leu Lys Ala 290 295 300 Ser
Arg Lys Leu Val Ala Glu His Pro Ile Glu Lys Val Gly Ala Glu 305 310
315 320 Leu Arg Ala Met Met Pro Trp Ile Ser Lys Asn Gln Leu Val Asp
Lys 325 330 335 Ala Lys Asn 451020DNASphingobium japonicum
45atgaaggttt attacgaccg cgacgccgac atcggcctga tcaagggcaa gaaggtcgcc
60atcctcggtt acggttcgca gggtcacgcc catgcgcaga atctgcgcga cagcggcgtc
120gccgaagtcg ccatcgcgct gcgccccggc tcgcccagcg ccaagaaggc
cgaaggcgcg 180ggcttcaagg tgctgccgaa cgcggaagcc gccgcatggg
ccgacgtgct gatgatcctg 240gcgcccgacg agcatcaggc cgccatctac
gccgccgaca tccacgccaa tctgcgcccc 300ggcgcggcgc tggccttcgc
gcacggcctc aacgtccatt tcggcctgat cgagccgcgc 360aaggatgtcg
acgtcatcat gatcgcgccc aagggtccgg gccacaccgt tcgcggcgaa
420tatgtgaagg gcggcggcgt gccctgcctg atcgccgttc atcaggacgc
gaccggcaac 480gcgcatgaca tcgccctgtc ctacgcttcg ggcgtcggcg
gcggccgcag cggcatcatc 540gaaaccaatt tccgcgagga atgcgaaacc
gacctgttcg gcgagcaggc cgtgctgtgc 600ggcggcgcga ccgcgctggt
ccaagcgggc ttcgaaacgc tggtcgaggc gggctacgcc 660cccgaaatgg
cctatttcga atgcctgcac gaactgaagc tgatcgtcga cctgatgtat
720gaaggcggca tcgccaacat gcgctattcg atctcgaaca ccgccgaata
tggcgatatc 780aagaccggcc cgcgcatcat caccgaagaa acgaagaagg
aaatgaagcg cgttctggcc 840gacatccagt cgggccgctt cgtcaaggac
ttcgtgctcg acaaccgcgc cggccagccg 900gaattgaagg ccagccgcat
cgccgcccag cgccacccga tcgaggaaac cggcgccaag 960ctgcgcgcca
tgatgccctg gatcggcgcg aacaagctgg tcgacaagga caggaactga
102046339PRTSphingobium japonicum 46Met Lys Val Tyr Tyr Asp Arg Asp
Ala Asp Ile Gly Leu Ile Lys Gly 1 5 10 15 Lys Lys Val Ala Ile Leu
Gly Tyr Gly Ser Gln Gly His Ala His Ala 20 25 30 Gln Asn Leu Arg
Asp Ser Gly Val Ala Glu Val Ala Ile Ala Leu Arg 35 40 45 Pro Gly
Ser Pro Ser Ala Lys Lys Ala Glu Gly Ala Gly Phe Lys Val 50 55 60
Leu Pro Asn Ala Glu Ala Ala Ala Trp Ala Asp Val Leu Met Ile Leu 65
70 75 80 Ala Pro Asp Glu His Gln Ala Ala Ile Tyr Ala Ala Asp Ile
His Ala 85 90 95 Asn Leu Arg Pro Gly Ala Ala Leu Ala Phe Ala His
Gly Leu Asn Val 100 105 110 His Phe Gly Leu Ile Glu Pro Arg Lys Asp
Val Asp Val Ile Met Ile 115 120 125 Ala Pro Lys Gly Pro Gly His Thr
Val Arg Gly Glu Tyr Val Lys Gly 130 135 140 Gly Gly Val Pro Cys Leu
Ile Ala Val His Gln Asp Ala Thr Gly Asn 145 150 155 160 Ala His Asp
Ile Ala Leu Ser Tyr Ala Ser Gly Val Gly Gly Gly Arg 165 170 175 Ser
Gly Ile Ile Glu Thr Asn Phe Arg Glu Glu Cys Glu Thr Asp Leu 180 185
190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Ala Thr Ala Leu Val Gln
195 200 205 Ala Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr Ala Pro Glu
Met Ala 210 215 220 Tyr Phe Glu Cys Leu His Glu Leu Lys Leu Ile Val
Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Ile Ala Asn Met Arg Tyr
Ser Ile Ser Asn Thr Ala Glu 245 250 255 Tyr Gly Asp Ile Lys Thr Gly
Pro Arg Ile Ile Thr Glu Glu Thr Lys 260 265 270 Lys Glu Met Lys Arg
Val Leu Ala Asp Ile Gln Ser Gly Arg Phe Val 275 280 285 Lys Asp Phe
Val Leu Asp Asn Arg Ala Gly Gln Pro Glu Leu Lys Ala 290 295 300 Ser
Arg Ile Ala Ala Gln Arg His Pro Ile Glu Glu Thr Gly Ala Lys 305 310
315 320 Leu Arg Ala Met Met Pro Trp Ile Gly Ala Asn Lys Leu Val Asp
Lys 325 330 335 Asp Arg Asn 471020DNAErythrobacter litoralis
47gtgaaagttt attacgacgc cgatgccgat cttggactga tcacggacaa gaagatcgcc
60gtgctcggct atggcagcca ggggcacgcc catgcacaga atctgcgtga cagcgggatc
120aaagaggtag caatcgcgct gcgcgacggc tcttccagcg cgaagaaagc
gcaggatgct 180ggcttcaagg tgctcagcaa ttccgacgct gccgagtggg
ccgatatcct gatgatcctc 240gcccccgacg agcaccaggc ggcgatctgg
gcggatgacc ttgcgggcaa catgaagccg 300ggcagcgccc tcgccttcgc
ccacgggctc aacatccact tcggcctgat cgaaccaccc 360gccgagatcg
acgtcatcat gatcgcgccg aagggtcctg gtcatactgt ccgcagcgag
420tatcagcgcg gcggcggcgt gccctgcctc atcgccgtcc accaggattc
gagcggcaat 480gccaaggaca tcgccctcgc ctatgccagc ggtgtcggcg
gcgggcgcag cggcattatc 540gagaccaact tccgcgagga atgcgagacc
gacctgttcg gcgagcaggc cgtgctgtgc 600ggcgggatca cgcacctgat
ccaagccggc ttcgagacgc tgaccgaggc cggatatgca 660ccggagatgg
cctatttcga gtgcctgcac gagaccaagc tgatcgtcga cctgctctac
720gaaggcggca tcgccaatat gcgctactcg atcagcaaca ccgccgagta
tggcgacatc 780accaccggcc cgcgcatcat caccgatgag accaaggccg
aaatgaagcg cgttctcggc 840gatatccagt cgggccgctt cgtgaagaac
ttcgtcctcg acaaccgcgc cggccagccc 900gaactcaagg ctgcccgcaa
gcgcgccgaa gcgcatccga tcgaacagac cggtgccaag 960ctgcgcgcaa
tgatgccgtg gatcggcaag aacaagctgg tcgacaagga caggaactag
102048339PRTErythrobacter litoralis 48Met Lys Val Tyr Tyr Asp Ala
Asp Ala Asp Leu Gly Leu Ile Thr Asp 1 5 10 15 Lys Lys Ile Ala Val
Leu Gly Tyr Gly Ser Gln Gly His Ala His Ala 20 25 30 Gln Asn Leu
Arg Asp Ser Gly Ile Lys Glu Val Ala Ile Ala Leu Arg 35 40 45 Asp
Gly Ser Ser Ser Ala Lys Lys Ala Gln Asp Ala Gly Phe Lys Val 50 55
60 Leu Ser Asn Ser Asp Ala Ala Glu Trp Ala Asp Ile Leu Met Ile Leu
65 70 75 80 Ala Pro Asp Glu His Gln Ala Ala Ile Trp Ala Asp Asp Leu
Ala Gly 85 90 95 Asn Met Lys Pro Gly Ser Ala Leu Ala Phe Ala His
Gly Leu Asn Ile 100 105 110 His Phe Gly Leu Ile Glu Pro Pro Ala Glu
Ile Asp Val Ile Met Ile 115 120 125 Ala Pro Lys Gly Pro Gly His Thr
Val Arg Ser Glu Tyr Gln Arg Gly 130 135 140 Gly Gly Val Pro Cys Leu
Ile Ala Val His Gln Asp Ser Ser Gly Asn 145 150 155 160 Ala Lys Asp
Ile Ala Leu Ala Tyr Ala Ser Gly Val Gly Gly Gly Arg 165 170 175 Ser
Gly Ile Ile Glu Thr Asn Phe Arg Glu Glu Cys Glu Thr Asp Leu 180 185
190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Ile Thr His Leu Ile Gln
195 200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Ala Pro Glu
Met Ala 210 215 220 Tyr Phe Glu Cys Leu His Glu Thr Lys Leu Ile Val
Asp Leu Leu Tyr 225 230 235 240 Glu Gly Gly Ile Ala Asn Met Arg Tyr
Ser Ile Ser Asn Thr Ala Glu 245 250 255 Tyr Gly Asp Ile Thr Thr Gly
Pro Arg Ile Ile Thr Asp Glu Thr Lys 260 265 270 Ala Glu Met Lys Arg
Val Leu Gly Asp Ile Gln Ser Gly Arg Phe Val 275 280 285 Lys Asn Phe
Val Leu Asp Asn Arg Ala Gly Gln Pro Glu Leu Lys Ala 290 295 300 Ala
Arg Lys Arg Ala Glu Ala His Pro Ile Glu Gln Thr Gly Ala Lys 305 310
315 320 Leu Arg Ala Met Met Pro Trp Ile Gly Lys Asn Lys Leu Val Asp
Lys 325 330 335 Asp Arg Asn 491020DNASphingobium chlorophenolicum
49atgaaggttt attacgaccg cgacgcagac atcggcctga tcaagggcaa gaaggtcgcc
60atcctgggtt acggttcgca gggtcacgcc catgcgcaga atctgcgcga ttccggcgtc
120gccgaagtcg ccatcgcgct gcgccccggc tcgccgagcg ccaagaaggc
cgaaggcgcg 180ggcttcaagg tgctggcgaa cgccgacgcc gccgcatggg
ccgatgtgct catgatcctg 240gcgcccgacg agcatcaggc cgccatctac
gccgacgaca tccacgccaa tctgcgcccc 300ggcgccgcgc tcgccttcgc
gcacggcctc aacgtgcatt tcggcctgat cgagccgcgc 360aaggacgtcg
acgtcatcat gatcgcgccc aagggccccg gccacaccgt gcgcggcgaa
420tatgtgaagg gcggcggcgt gccctgcctg atcgccatcc atcaggacgc
gaccggcaac 480gcccatgaca tcgccctgtc ctacgcttcg ggcgtcggcg
gcggccgcag cggcatcatc 540gaaaccaact tccgcgagga atgcgaaacc
gacctgttcg gcgagcaggc cgtgctgtgc 600ggcggcgcca ccgcgctggt
gcaggcgggc ttcgaaacgc tggtcgaggc tggctacgcc 660ccggaaatgg
cctatttcga atgcctgcac gaactgaagc tgatcgtcga cctgatgtat
720gaaggcggca tcgccaacat gcgctattcg atctcgaaca ccgccgaata
tggcgatatc 780aagaccggcc cgcgcatcat caccgatgaa acgaagaagg
aaatgaagcg cgttctggcc 840gacatccagt cgggccgctt cgtcaaggac
ttcgtgctcg acaaccgcgc cggccagccg 900gaattgaagg ccagccgcat
cgccgcccag cgccacccga tcgaggaaac cggcgccaag 960ctgcgcgcca
tgatgccctg gatcggcgcg aacaagctgg tcgacaagga caagaactga
102050339PRTSphingobium chlorophenolicum 50Met Lys Val Tyr Tyr Asp
Arg Asp Ala Asp Ile Gly Leu Ile Lys Gly 1 5 10 15 Lys Lys Val Ala
Ile Leu Gly Tyr Gly Ser Gln Gly His Ala His Ala 20 25 30 Gln Asn
Leu Arg Asp Ser Gly Val Ala Glu Val Ala Ile Ala Leu Arg 35 40 45
Pro Gly Ser Pro Ser Ala Lys Lys Ala Glu Gly Ala Gly Phe Lys Val 50
55 60 Leu Ala Asn Ala Asp Ala Ala Ala Trp Ala Asp Val Leu Met Ile
Leu 65 70 75 80 Ala Pro Asp Glu His Gln Ala Ala Ile Tyr Ala Asp Asp
Ile His Ala 85 90 95 Asn Leu Arg Pro Gly Ala Ala Leu Ala Phe Ala
His Gly Leu Asn Val 100 105 110 His Phe Gly Leu Ile Glu Pro Arg Lys
Asp Val Asp Val Ile Met Ile 115 120 125 Ala Pro Lys Gly Pro Gly His
Thr Val Arg Gly Glu Tyr Val Lys Gly 130 135 140 Gly Gly Val Pro Cys
Leu Ile Ala Ile His Gln Asp Ala Thr Gly Asn 145 150 155 160 Ala His
Asp Ile Ala Leu Ser Tyr Ala Ser Gly Val Gly Gly Gly Arg 165 170 175
Ser Gly Ile Ile Glu Thr Asn Phe Arg Glu Glu Cys Glu Thr Asp Leu 180
185 190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Ala Thr Ala Leu Val
Gln 195 200 205 Ala Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr Ala Pro
Glu Met Ala 210 215 220 Tyr Phe Glu Cys Leu His Glu Leu Lys Leu Ile
Val Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Ile Ala Asn Met Arg
Tyr Ser Ile Ser Asn Thr Ala Glu 245 250 255 Tyr Gly Asp Ile Lys Thr
Gly Pro Arg Ile Ile Thr Asp Glu Thr Lys 260 265 270 Lys Glu Met Lys
Arg Val Leu Ala Asp Ile Gln Ser Gly Arg Phe Val 275 280 285 Lys Asp
Phe Val Leu Asp Asn Arg Ala Gly Gln Pro Glu Leu Lys Ala 290 295 300
Ser Arg Ile Ala Ala Gln Arg His Pro Ile Glu Glu Thr Gly Ala Lys 305
310 315 320 Leu Arg Ala Met Met Pro Trp Ile Gly Ala Asn Lys Leu Val
Asp Lys 325 330 335 Asp Lys Asn 511020DNASphingomonas sp.
51atgcgtgtct attatgatcg cgacgccgat ctgaacctga tctccgagaa gaacatcgcc
60atcctgggtt acggctcgca gggccatgcc catgcgcaga acctgcgcga ttcgggcgtc
120aagaatgtcg cgatcgcgct gcgccccggt tcggcctcgg ccgccaaggc
ggaggctgcg 180ggcttcaagg tcctgtcgaa caaggaagcg gccggctggg
ccgacatcct gatgatcctg 240gcccccgacg agcatcaggc cgcgatctat
gacgccgacc tgaagggcaa tttgaagccg 300ggcgccgcgc tcgccttcgc
gcacggcctg aacgtgcatt tcggcctgat cgagccgcct 360gcggacatcg
acgttatcat gatcgcgccg aagggccccg gccacaccgt gcgcagcgaa
420tatgtgcgcg gcggcggcgt gccctgcctg atcgcgatcc atcaggacgc
cagcggcaac 480gcgcatgacg tggcgctggc ctatgcgtcg ggcgtcggcg
gcggtcgctc gggcatcatc 540gagacgaact tccgcgaaga gtgcgaaacc
gatctgttcg gcgagcaggc cgtgctgtgt 600ggcggcgcga ccgcgctggt
ccaggcgggc ttcgagacgc tggtcgaggc gggctatgcc 660cccgaaatgg
cgtatttcga gtgcctccac gagctgaagc tgatcgtcga cctgatgtat
720gagggcggca tcgccaacat gcgctactcg atctcgaaca ccgccgagta
cggcgacatc 780aagaccggcc cacgcatcat cactgaagag accaagaagg
aaatgaagcg cgtgctcgcc 840gacatccagt cgggccgctt cgtgaaggac
ttcgtgctcg acaaccgcgc cggccagccc 900gaactgaagg ccagccgcat
cgccgccaag cgccatcaga tcgaacaggt cggcagcgaa 960ctgcgcgcga
tgatgccgtg gatcggcgcg aacaagctgg tggacaaggc gaagaactga
102052339PRTSphingomonas sp. 52Met Arg Val Tyr Tyr Asp Arg Asp Ala
Asp Leu Asn Leu Ile Ser Glu 1 5 10 15 Lys Asn Ile Ala Ile Leu Gly
Tyr Gly Ser Gln Gly His Ala His Ala 20 25 30 Gln Asn Leu Arg Asp
Ser Gly Val Lys Asn Val Ala Ile Ala Leu Arg 35 40 45 Pro Gly Ser
Ala Ser Ala Ala Lys Ala Glu Ala Ala Gly Phe Lys Val 50 55 60 Leu
Ser Asn Lys Glu Ala Ala Gly Trp Ala Asp Ile Leu Met Ile Leu 65 70
75 80 Ala Pro Asp Glu His Gln Ala Ala Ile Tyr Asp Ala Asp Leu Lys
Gly 85 90 95 Asn Leu Lys Pro Gly Ala Ala Leu Ala Phe Ala His Gly
Leu Asn Val 100 105 110 His Phe Gly Leu Ile Glu Pro Pro Ala Asp Ile
Asp Val Ile Met Ile 115 120
125 Ala Pro Lys Gly Pro Gly His Thr Val Arg Ser Glu Tyr Val Arg Gly
130 135 140 Gly Gly Val Pro Cys Leu Ile Ala Ile His Gln Asp Ala Ser
Gly Asn 145 150 155 160 Ala His Asp Val Ala Leu Ala Tyr Ala Ser Gly
Val Gly Gly Gly Arg 165 170 175 Ser Gly Ile Ile Glu Thr Asn Phe Arg
Glu Glu Cys Glu Thr Asp Leu 180 185 190 Phe Gly Glu Gln Ala Val Leu
Cys Gly Gly Ala Thr Ala Leu Val Gln 195 200 205 Ala Gly Phe Glu Thr
Leu Val Glu Ala Gly Tyr Ala Pro Glu Met Ala 210 215 220 Tyr Phe Glu
Cys Leu His Glu Leu Lys Leu Ile Val Asp Leu Met Tyr 225 230 235 240
Glu Gly Gly Ile Ala Asn Met Arg Tyr Ser Ile Ser Asn Thr Ala Glu 245
250 255 Tyr Gly Asp Ile Lys Thr Gly Pro Arg Ile Ile Thr Glu Glu Thr
Lys 260 265 270 Lys Glu Met Lys Arg Val Leu Ala Asp Ile Gln Ser Gly
Arg Phe Val 275 280 285 Lys Asp Phe Val Leu Asp Asn Arg Ala Gly Gln
Pro Glu Leu Lys Ala 290 295 300 Ser Arg Ile Ala Ala Lys Arg His Gln
Ile Glu Gln Val Gly Ser Glu 305 310 315 320 Leu Arg Ala Met Met Pro
Trp Ile Gly Ala Asn Lys Leu Val Asp Lys 325 330 335 Ala Lys Asn
531020DNANovosphingobium nitrogenifigens 53atgaaggttt actacgacgc
cgacgccgat ctcaacctga tcaccgggaa gaaggtcgcc 60atcctgggct atggcagcca
gggccacgcc cacgcgcaga acctgcgcga ttcgggcgtc 120aaggaagtgg
cgatcgcgct gcgtcccggt tcggccagcg ccgccaaggc tgaaggcgcc
180ggtttcaagg tcatggcgaa cgccgaagcc gctgcctggg ccgacgttct
catgattctc 240gcgcccgacg aacatcaggc cgcgatctat gccgacgaca
tccacgccaa cctgcgcccc 300ggcgcggcgc tggccttcgc acacggcctc
aacgttcact tcggcctgat cgaaccgcgc 360gccgacgtcg acgtgatcat
gatcgcgccc aagggcccgg gccacaccgt tcgcggtgaa 420tacgtgaagg
gcggcggggt gccctgcctc atcgccatcg cgcaggacgc gaccggcaat
480gcccacgaca tcgcccttgc ctatgcttcg ggtgtcggcg gcggccgttc
gggtatcatc 540gaaaccaact tcaaggaaga gtgcgaaacc gacctgttcg
gcgaacaagc cgttctttgc 600ggcggcctga cccacctcat ccaggctggt
ttcgaaaccc tggtcgaagc cggttacgcg 660ccggaaatgg cctatttcga
atgcctccac gaagtgaagc tgatcgtcga cctgatgtat 720gaaggcggca
tcgccaacat gcgctactcg atctcgaaca cggccgaata cggtgacatc
780accactggtc cgcgcctgat caccgccgaa accaaggcgg aaatgaagcg
cgtcctcgaa 840gacatccagg ccggtcgctt cgtcaagaac ttcgtgctcg
acaaccgcgc tggccagccc 900gagctgaagg ctgcccgcaa ggctgccgct
gcgcacccga tcgaacagac cggcgctcgc 960ctgcgtgcga tgatgccctg
gatcggtgcg aaccagctgg tcgacaaggc caagaactga
102054339PRTNovosphingobium nitrogenifigens 54Met Lys Val Tyr Tyr
Asp Ala Asp Ala Asp Leu Asn Leu Ile Thr Gly 1 5 10 15 Lys Lys Val
Ala Ile Leu Gly Tyr Gly Ser Gln Gly His Ala His Ala 20 25 30 Gln
Asn Leu Arg Asp Ser Gly Val Lys Glu Val Ala Ile Ala Leu Arg 35 40
45 Pro Gly Ser Ala Ser Ala Ala Lys Ala Glu Gly Ala Gly Phe Lys Val
50 55 60 Met Ala Asn Ala Glu Ala Ala Ala Trp Ala Asp Val Leu Met
Ile Leu 65 70 75 80 Ala Pro Asp Glu His Gln Ala Ala Ile Tyr Ala Asp
Asp Ile His Ala 85 90 95 Asn Leu Arg Pro Gly Ala Ala Leu Ala Phe
Ala His Gly Leu Asn Val 100 105 110 His Phe Gly Leu Ile Glu Pro Arg
Ala Asp Val Asp Val Ile Met Ile 115 120 125 Ala Pro Lys Gly Pro Gly
His Thr Val Arg Gly Glu Tyr Val Lys Gly 130 135 140 Gly Gly Val Pro
Cys Leu Ile Ala Ile Ala Gln Asp Ala Thr Gly Asn 145 150 155 160 Ala
His Asp Ile Ala Leu Ala Tyr Ala Ser Gly Val Gly Gly Gly Arg 165 170
175 Ser Gly Ile Ile Glu Thr Asn Phe Lys Glu Glu Cys Glu Thr Asp Leu
180 185 190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Thr His Leu
Ile Gln 195 200 205 Ala Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr Ala
Pro Glu Met Ala 210 215 220 Tyr Phe Glu Cys Leu His Glu Val Lys Leu
Ile Val Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Ile Ala Asn Met
Arg Tyr Ser Ile Ser Asn Thr Ala Glu 245 250 255 Tyr Gly Asp Ile Thr
Thr Gly Pro Arg Leu Ile Thr Ala Glu Thr Lys 260 265 270 Ala Glu Met
Lys Arg Val Leu Glu Asp Ile Gln Ala Gly Arg Phe Val 275 280 285 Lys
Asn Phe Val Leu Asp Asn Arg Ala Gly Gln Pro Glu Leu Lys Ala 290 295
300 Ala Arg Lys Ala Ala Ala Ala His Pro Ile Glu Gln Thr Gly Ala Arg
305 310 315 320 Leu Arg Ala Met Met Pro Trp Ile Gly Ala Asn Gln Leu
Val Asp Lys 325 330 335 Ala Lys Asn 551080DNABacteroides
thetaiotaomicron 55atggcgcaag tcatcaaaac aaaaaaacaa aaaaaaatgg
cacagttgaa ttttggcgga 60actgtagaaa atgtagttat ccgtgatgaa tttccattgg
aaaaagctcg tgaagtattg 120aaaaatgaaa caatcgctgt aatcggttat
ggcgtacaag gtcctggaca ggctctgaac 180cttcgtgata acggtttcaa
tgtaatcgtt ggtcaacgcc agggaaagac atatgacaaa 240gcggtagctg
acggatgggt tccgggtgaa actttgttcg gtattgaaga agcttgcgaa
300aaaggtacga tcattatgtg cctgttgtct gatgcagcgg taatgtctgt
atggcctact 360atcaagcctt acctgactgc aggaaaagct ctttatttct
ctcatggttt tgctattaca 420tggagtgatc gcacaggtgt agttcctcct
gcagatatcg acgtaatcat ggttgctcct 480aaaggttcgg gtacatcctt
gcgtactatg ttccttgaag gtcgcggctt gaactcttct 540tacgctatct
atcaggatgc aacaggcaac gctatggaca gaacaatcgc attgggtatc
600ggtatcggtt caggttattt gttcgaaaca actttcatcc gcgaagctac
ttccgacctg 660acaggcgaac gtggttcatt gatgggagct atccagggtc
tgttgctggc acaatacgaa 720gtgttacgtg aaaacggtca cactccttcc
gaagcattca acgaaactgt agaagagctg 780actcagtcat tgatgccgtt
gtttgcaaag aacggtatgg actggatgta tgctaactgc 840tctactacag
ctcaacgtgg tgctctcgac tggatgggcc ccttccacga tgctatcaaa
900ccggtagttg aaaagttgta tcacagtgtg aagactggta acgaagcaca
gatttcaatc 960gactctaact ccaaaccgga ttatcgtgag aaactggaag
aagaactgaa agcattgcgc 1020gaaagcgaaa tgtggcagac tgccgtgaca
gttcgtaaac ttcgtccgga aaataattaa 108056359PRTBacteroides
thetaiotaomicron 56Met Ala Gln Val Ile Lys Thr Lys Lys Gln Lys Lys
Met Ala Gln Leu 1 5 10 15 Asn Phe Gly Gly Thr Val Glu Asn Val Val
Ile Arg Asp Glu Phe Pro 20 25 30 Leu Glu Lys Ala Arg Glu Val Leu
Lys Asn Glu Thr Ile Ala Val Ile 35 40 45 Gly Tyr Gly Val Gln Gly
Pro Gly Gln Ala Leu Asn Leu Arg Asp Asn 50 55 60 Gly Phe Asn Val
Ile Val Gly Gln Arg Gln Gly Lys Thr Tyr Asp Lys 65 70 75 80 Ala Val
Ala Asp Gly Trp Val Pro Gly Glu Thr Leu Phe Gly Ile Glu 85 90 95
Glu Ala Cys Glu Lys Gly Thr Ile Ile Met Cys Leu Leu Ser Asp Ala 100
105 110 Ala Val Met Ser Val Trp Pro Thr Ile Lys Pro Tyr Leu Thr Ala
Gly 115 120 125 Lys Ala Leu Tyr Phe Ser His Gly Phe Ala Ile Thr Trp
Ser Asp Arg 130 135 140 Thr Gly Val Val Pro Pro Ala Asp Ile Asp Val
Ile Met Val Ala Pro 145 150 155 160 Lys Gly Ser Gly Thr Ser Leu Arg
Thr Met Phe Leu Glu Gly Arg Gly 165 170 175 Leu Asn Ser Ser Tyr Ala
Ile Tyr Gln Asp Ala Thr Gly Asn Ala Met 180 185 190 Asp Arg Thr Ile
Ala Leu Gly Ile Gly Ile Gly Ser Gly Tyr Leu Phe 195 200 205 Glu Thr
Thr Phe Ile Arg Glu Ala Thr Ser Asp Leu Thr Gly Glu Arg 210 215 220
Gly Ser Leu Met Gly Ala Ile Gln Gly Leu Leu Leu Ala Gln Tyr Glu 225
230 235 240 Val Leu Arg Glu Asn Gly His Thr Pro Ser Glu Ala Phe Asn
Glu Thr 245 250 255 Val Glu Glu Leu Thr Gln Ser Leu Met Pro Leu Phe
Ala Lys Asn Gly 260 265 270 Met Asp Trp Met Tyr Ala Asn Cys Ser Thr
Thr Ala Gln Arg Gly Ala 275 280 285 Leu Asp Trp Met Gly Pro Phe His
Asp Ala Ile Lys Pro Val Val Glu 290 295 300 Lys Leu Tyr His Ser Val
Lys Thr Gly Asn Glu Ala Gln Ile Ser Ile 305 310 315 320 Asp Ser Asn
Ser Lys Pro Asp Tyr Arg Glu Lys Leu Glu Glu Glu Leu 325 330 335 Lys
Ala Leu Arg Glu Ser Glu Met Trp Gln Thr Ala Val Thr Val Arg 340 345
350 Lys Leu Arg Pro Glu Asn Asn 355 571215DNASchizosaccharomyces
pombe 57atgtctttcc gtaattcctc tagaatggcc atgaaggcct tgcgtactat
gggtagccgt 60cgtttggcta ctcgtagcat gtctgttatg gctcgcacca ttgctgcccc
cagcatgcgt 120tttgcgcctc gcatgaccgc ccctttgatg caaactcgcg
gtatgcgtgt tatggacttt 180gccggtacca aggagaacgt ttgggagcgt
tctgactggc ctcgtgaaaa gcttgttgac 240tacttcaaga acgacactct
tgccatcatt ggatacggat ctcaaggaca tggtcaaggt 300ttgaacgctc
gtgatcaagg tttgaacgtt attgtcggtg tccgtaagga tggtgcttcc
360tggaagcaag ctattgaaga cggttgggtc cctggtaaga ctttgttccc
cgtcgaggag 420gccatcaaga agggttctat catcatgaac cttttgtccg
atgccgctca aactgagact 480tggcccaaga ttgctcccct tattaccaag
ggtaagactt tgtacttctc tcacggtttc 540tccgtcatct tcaaggatca
aactaagatt caccccccta aggatgttga tgttatcctt 600gtcgctccca
agggttctgg tcgtaccgtt cgtacccttt tcaaggaagg tcgtggtatt
660aactcttcct tcgctgttta ccaagacgtt actggtaagg ctcaagaaaa
ggccattggt 720ttggctgttg ccgtcggttc cggtttcatc taccaaacca
ctttcaagaa ggaggttatc 780tccgatttgg ttggtgagcg tggatgtctc
atgggtggta tcaacggtct tttcttggct 840caataccaag ttttgcgtga
acgtggtcac tcccctgctg aggctttcaa cgagactgtt 900gaagaggcca
ctcaatccct ttaccccttg attggcaagt acggtcttga ctacatgttt
960gccgcttgct ctaccaccgc tcgtcgtggt gccattgact ggactcctcg
tttccttgag 1020gctaacaaga aggtccttaa tgaattgtat gacaatgttg
agaacggtaa cgaggctaag 1080cgttccttgg aatacaactc tgctcccaac
taccgtgagc tttacgataa ggagttggag 1140gaaatccgca acttggaaat
ctggaaggct ggtgaggttg ttcgttctct ccgtcctgaa 1200cacaacaagc actag
121558404PRTSchizosaccharomyces pombe 58Met Ser Phe Arg Asn Ser Ser
Arg Met Ala Met Lys Ala Leu Arg Thr 1 5 10 15 Met Gly Ser Arg Arg
Leu Ala Thr Arg Ser Met Ser Val Met Ala Arg 20 25 30 Thr Ile Ala
Ala Pro Ser Met Arg Phe Ala Pro Arg Met Thr Ala Pro 35 40 45 Leu
Met Gln Thr Arg Gly Met Arg Val Met Asp Phe Ala Gly Thr Lys 50 55
60 Glu Asn Val Trp Glu Arg Ser Asp Trp Pro Arg Glu Lys Leu Val Asp
65 70 75 80 Tyr Phe Lys Asn Asp Thr Leu Ala Ile Ile Gly Tyr Gly Ser
Gln Gly 85 90 95 His Gly Gln Gly Leu Asn Ala Arg Asp Gln Gly Leu
Asn Val Ile Val 100 105 110 Gly Val Arg Lys Asp Gly Ala Ser Trp Lys
Gln Ala Ile Glu Asp Gly 115 120 125 Trp Val Pro Gly Lys Thr Leu Phe
Pro Val Glu Glu Ala Ile Lys Lys 130 135 140 Gly Ser Ile Ile Met Asn
Leu Leu Ser Asp Ala Ala Gln Thr Glu Thr 145 150 155 160 Trp Pro Lys
Ile Ala Pro Leu Ile Thr Lys Gly Lys Thr Leu Tyr Phe 165 170 175 Ser
His Gly Phe Ser Val Ile Phe Lys Asp Gln Thr Lys Ile His Pro 180 185
190 Pro Lys Asp Val Asp Val Ile Leu Val Ala Pro Lys Gly Ser Gly Arg
195 200 205 Thr Val Arg Thr Leu Phe Lys Glu Gly Arg Gly Ile Asn Ser
Ser Phe 210 215 220 Ala Val Tyr Gln Asp Val Thr Gly Lys Ala Gln Glu
Lys Ala Ile Gly 225 230 235 240 Leu Ala Val Ala Val Gly Ser Gly Phe
Ile Tyr Gln Thr Thr Phe Lys 245 250 255 Lys Glu Val Ile Ser Asp Leu
Val Gly Glu Arg Gly Cys Leu Met Gly 260 265 270 Gly Ile Asn Gly Leu
Phe Leu Ala Gln Tyr Gln Val Leu Arg Glu Arg 275 280 285 Gly His Ser
Pro Ala Glu Ala Phe Asn Glu Thr Val Glu Glu Ala Thr 290 295 300 Gln
Ser Leu Tyr Pro Leu Ile Gly Lys Tyr Gly Leu Asp Tyr Met Phe 305 310
315 320 Ala Ala Cys Ser Thr Thr Ala Arg Arg Gly Ala Ile Asp Trp Thr
Pro 325 330 335 Arg Phe Leu Glu Ala Asn Lys Lys Val Leu Asn Glu Leu
Tyr Asp Asn 340 345 350 Val Glu Asn Gly Asn Glu Ala Lys Arg Ser Leu
Glu Tyr Asn Ser Ala 355 360 365 Pro Asn Tyr Arg Glu Leu Tyr Asp Lys
Glu Leu Glu Glu Ile Arg Asn 370 375 380 Leu Glu Ile Trp Lys Ala Gly
Glu Val Val Arg Ser Leu Arg Pro Glu 385 390 395 400 His Asn Lys His
591215DNASchizosaccharomyces pombe 59atgtctttcc gtaattcctc
tagaatggcc atgaaggcct tgcgtactat gggtagccga 60cgtttggcta ctcgtagcat
gtctgttatg gctcgcacca ttgctgcccc cagaatgcgt 120tgggcgcctc
gcatgaccgc ccctttgatg caaactcgcg gtatgcgtgt tatggacttt
180gccggtacca aggagaacgt ttgggagcgc tctgactggc ctcgtgaaaa
gcttgttgac 240tacttcaaga acgacactct tgccatcatt ggatccggat
ctcaaggaca tggtcaaggt 300ttgaacgctc gtgatcaagg tttgaacgtt
attgtcggtg tccgtaagga tggtgcttcc 360tggaagcaag ctattgaaga
cggttgggtc cctggtaaga ctttgttccc cgtcgaggag 420gccatcaaga
agggttctat catcatgaac cttttgtccg atgccgctca aactgagact
480tggcccaaga ttgctcccct tattaccaag ggtaagactt tgtacttctc
tcacggtttc 540tccgtcatct tcaaggatca aactaagatt caccccccta
aggatgttga tgttatcctt 600gtcgctccca agggttctgg tcgtaccgtt
cgtacccttt tcaaggaagg tcgtggtatt 660aactcttcct tcgctgttta
ccaagacgtt actggtaagg ctcaagaaaa gaccattggt 720ttggctgttg
ccgtcggttc cggtttcatc taccaaacca ctttcaagaa ggaggttatc
780tccgatttgg ttggtgagcg tggatgtctc atgggtggta tccccggtct
tttcttggct 840caataccaag ttttgcgtga acgtggtcac tcccctgctg
aggctttccc cgagactgtt 900gaagaggcca ctcaatccct ttaccccttg
attggcaagt acggtcttga ctacatgttt 960gccgcttgct ctaccaccgc
tcgtcgtggt gccattgact ggactcctcg tttccttgag 1020gctaacaaga
aggtccttaa tgaattgtat gacaatgttg agaacggtaa cgaggctaag
1080cgttccttgg aatacaactc tgctcccaac taccgtgagc tttacgataa
ggagttggag 1140gaaatccgca acttggaaat ctggaaggct ggtgaggttg
gtcggtctct ccggcctgaa 1200cacaacaagc actag
121560404PRTSchizosaccharomyces pombe 60Met Ser Phe Arg Asn Ser Ser
Arg Met Ala Met Lys Ala Leu Arg Thr 1 5 10 15 Met Gly Ser Arg Arg
Leu Ala Thr Arg Ser Met Ser Val Met Ala Arg 20 25 30 Thr Ile Ala
Ala Pro Arg Met Arg Trp Ala Pro Arg Met Thr Ala Pro 35 40 45 Leu
Met Gln Thr Arg Gly Met Arg Val Met Asp Phe Ala Gly Thr Lys 50 55
60 Glu Asn Val Trp Glu Arg Ser Asp Trp Pro Arg Glu Lys Leu Val Asp
65 70 75 80 Tyr Phe Lys Asn Asp Thr Leu Ala Ile Ile Gly Ser Gly Ser
Gln Gly 85 90 95 His Gly Gln Gly Leu Asn Ala Arg Asp Gln Gly Leu
Asn Val Ile Val 100 105 110 Gly Val Arg Lys Asp Gly Ala Ser Trp Lys
Gln Ala Ile Glu Asp Gly 115 120 125 Trp Val Pro Gly Lys Thr Leu Phe
Pro Val Glu Glu Ala Ile Lys Lys 130 135 140 Gly Ser Ile Ile Met Asn
Leu Leu Ser Asp Ala Ala Gln Thr Glu Thr 145 150 155 160 Trp Pro Lys
Ile Ala Pro Leu Ile Thr Lys Gly Lys Thr Leu Tyr Phe 165 170 175 Ser
His Gly Phe Ser Val Ile Phe Lys Asp Gln Thr Lys Ile His Pro 180 185
190 Pro Lys Asp Val Asp Val Ile Leu Val Ala Pro Lys Gly Ser Gly Arg
195 200 205 Thr Val Arg Thr Leu Phe Lys Glu Gly Arg Gly Ile Asn Ser
Ser Phe 210 215 220 Ala Val Tyr Gln Asp Val Thr Gly Lys Ala Gln Glu
Lys Thr Ile Gly 225 230 235
240 Leu Ala Val Ala Val Gly Ser Gly Phe Ile Tyr Gln Thr Thr Phe Lys
245 250 255 Lys Glu Val Ile Ser Asp Leu Val Gly Glu Arg Gly Cys Leu
Met Gly 260 265 270 Gly Ile Pro Gly Leu Phe Leu Ala Gln Tyr Gln Val
Leu Arg Glu Arg 275 280 285 Gly His Ser Pro Ala Glu Ala Phe Pro Glu
Thr Val Glu Glu Ala Thr 290 295 300 Gln Ser Leu Tyr Pro Leu Ile Gly
Lys Tyr Gly Leu Asp Tyr Met Phe 305 310 315 320 Ala Ala Cys Ser Thr
Thr Ala Arg Arg Gly Ala Ile Asp Trp Thr Pro 325 330 335 Arg Phe Leu
Glu Ala Asn Lys Lys Val Leu Asn Glu Leu Tyr Asp Asn 340 345 350 Val
Glu Asn Gly Asn Glu Ala Lys Arg Ser Leu Glu Tyr Asn Ser Ala 355 360
365 Pro Asn Tyr Arg Glu Leu Tyr Asp Lys Glu Leu Glu Glu Ile Arg Asn
370 375 380 Leu Glu Ile Trp Lys Ala Gly Glu Val Gly Arg Ser Leu Arg
Pro Glu 385 390 395 400 His Asn Lys His
611212DNASchizosaccharomyces japonicus 61atgtctttcc gttccgcctc
taagttggcc atgaaggcct tccgccaaaa tggtgcccgc 60cgcgttatcc ctgctacccg
ctctatgtct gtcttggctc gcgccggcgt tatgagccgc 120tctgctgctc
gtgttgcccc catggtccaa acccgtggtg tccgtactat ggactttggc
180ggtgttaagg agaccgtctg ggagcgtaac gactggcccc gtgagaagct
tcttgactac 240ttcaagaacg acactcttgc cgttattggt tacggttccc
aaggtcacgg acaaggtttg 300aacgctcgtg acaacggttt gaacgttatt
gttggtgttc gtgagggtgg tgcctcctgg 360aaggctgcca tcgaggacgg
ttgggtcccc ggcaagaact tgttccccat ggaggaggcc 420atcaagaagg
gtaccatcat catggacctt ctttccgatg ccgctcaaac cgagacctgg
480cccactattg ctccccttct caccaagggt aagactctct acttctctca
cggtttctcc 540gtcgtcttca aggaccaaac caaggttgtc ccccctaagg
acatcgatgt catccttgct 600gcccccaagg gttccggccg taccgtccgt
tccttgttca aggagggccg tggtatcaac 660tcctccgtcg ccgtcttcca
aaacgtttcc ggcaaggctg acgagaaggc tgttgctatt 720gctgtcgcca
ttggctccgg tttcatctac aagaccacct tcgagcgcga ggtcgtttct
780gacttggtcg gtgagcgtgg ttgccttatg ggtggtatca acggtctctt
cttggcccaa 840taccaaactc tccgtgagca cggccacacc cccgccgagg
ccttcaacga gactgttgag 900gaggccactc aatcccttta ccccttgatc
ggtaagtacg gtttggacta catgttcgcc 960gcctgctcta ccactgctcg
tcgtggtgcc attgactgga ctcaacgctt ctacgacgcc 1020aacaagaagg
tccttgagga cttgtacgag aacgttgcca acggtaacga ggccaagcgc
1080tccttggagt acaactctaa gcccaactac cgtgagcttt acgagaagga
gctcgctgag 1140atccgcgact tggagatctg gagagccggt gagactgtcc
gttctctccg tcccgaggag 1200aacaagcact ag
121262403PRTSchizosaccharomyces japonicus 62Met Ser Phe Arg Ser Ala
Ser Lys Leu Ala Met Lys Ala Phe Arg Gln 1 5 10 15 Asn Gly Ala Arg
Arg Val Ile Pro Ala Thr Arg Ser Met Ser Val Leu 20 25 30 Ala Arg
Ala Gly Val Met Ser Arg Ser Ala Ala Arg Val Ala Pro Met 35 40 45
Val Gln Thr Arg Gly Val Arg Thr Met Asp Phe Gly Gly Val Lys Glu 50
55 60 Thr Val Trp Glu Arg Asn Asp Trp Pro Arg Glu Lys Leu Leu Asp
Tyr 65 70 75 80 Phe Lys Asn Asp Thr Leu Ala Val Ile Gly Tyr Gly Ser
Gln Gly His 85 90 95 Gly Gln Gly Leu Asn Ala Arg Asp Asn Gly Leu
Asn Val Ile Val Gly 100 105 110 Val Arg Glu Gly Gly Ala Ser Trp Lys
Ala Ala Ile Glu Asp Gly Trp 115 120 125 Val Pro Gly Lys Asn Leu Phe
Pro Met Glu Glu Ala Ile Lys Lys Gly 130 135 140 Thr Ile Ile Met Asp
Leu Leu Ser Asp Ala Ala Gln Thr Glu Thr Trp 145 150 155 160 Pro Thr
Ile Ala Pro Leu Leu Thr Lys Gly Lys Thr Leu Tyr Phe Ser 165 170 175
His Gly Phe Ser Val Val Phe Lys Asp Gln Thr Lys Val Val Pro Pro 180
185 190 Lys Asp Ile Asp Val Ile Leu Ala Ala Pro Lys Gly Ser Gly Arg
Thr 195 200 205 Val Arg Ser Leu Phe Lys Glu Gly Arg Gly Ile Asn Ser
Ser Val Ala 210 215 220 Val Phe Gln Asn Val Ser Gly Lys Ala Asp Glu
Lys Ala Val Ala Ile 225 230 235 240 Ala Val Ala Ile Gly Ser Gly Phe
Ile Tyr Lys Thr Thr Phe Glu Arg 245 250 255 Glu Val Val Ser Asp Leu
Val Gly Glu Arg Gly Cys Leu Met Gly Gly 260 265 270 Ile Asn Gly Leu
Phe Leu Ala Gln Tyr Gln Thr Leu Arg Glu His Gly 275 280 285 His Thr
Pro Ala Glu Ala Phe Asn Glu Thr Val Glu Glu Ala Thr Gln 290 295 300
Ser Leu Tyr Pro Leu Ile Gly Lys Tyr Gly Leu Asp Tyr Met Phe Ala 305
310 315 320 Ala Cys Ser Thr Thr Ala Arg Arg Gly Ala Ile Asp Trp Thr
Gln Arg 325 330 335 Phe Tyr Asp Ala Asn Lys Lys Val Leu Glu Asp Leu
Tyr Glu Asn Val 340 345 350 Ala Asn Gly Asn Glu Ala Lys Arg Ser Leu
Glu Tyr Asn Ser Lys Pro 355 360 365 Asn Tyr Arg Glu Leu Tyr Glu Lys
Glu Leu Ala Glu Ile Arg Asp Leu 370 375 380 Glu Ile Trp Arg Ala Gly
Glu Thr Val Arg Ser Leu Arg Pro Glu Glu 385 390 395 400 Asn Lys His
631476DNASalmonella enterica 63atggctaact actttaatac actgaatctg
cgccagcagc tggcgcagct gggtaaatgc 60cgctttatgg gccgcgacga attcgccgac
ggcgcgagct accttcaggg taaaaaagtg 120gtcatcgtcg gctgtggcgc
tcaggggctg aaccagggcc tgaacatgcg tgactccggt 180ctggatattt
cctatgccct gcgtaaagaa gccattgctg agaagcgtgc ctcctggcgt
240aaagcgaccg aaaacggctt caaagtgggt acctacgaag agctgattcc
gcaggctgac 300ctggtggtta acctgacgcc ggacaaacag cactccgacg
tggtgcgctc cgtacagccg 360ctgatgaaag acggcgcggc gctgggctac
tcccacggct tcaatatcgt ggaggtgggc 420gagcagatcc gtaaagacat
caccgtggtg atggtagcgc cgaagtgtcc gggcaccgaa 480gtgcgcgaag
agtacaaacg tggtttcggc gtgccgacgc tgatcgccgt tcacccggaa
540aacgatccga aaggcgaagg catggcgatt gctaaagcct gggcagcagc
aactggcggt 600caccgtgcgg gcgtactgga atcttctttc gtggcggaag
tgaaatccga cctgatgggc 660gagcagacta tcctgtgcgg tatgctgcag
gctggttctc tgctgtgctt cgacaagctg 720gtggcagaag gcaccgaccc
ggcatacgcc gaaaaactga ttcagttcgg ctgggaaacc 780atcaccgaag
cgctgaagca gggcggcatc accctgatga tggaccgtct gtctaacccg
840gcgaaactgc gtgcttacgc gctgtccgaa cagctgaaag agatcatggc
gccgctgttc 900cagaaacaca tggatgacat catctccggc gagttctctt
ccggcatgat ggctgactgg 960gctaacgacg ataagaaact gctgacctgg
cgtgaagaga ccggtaaaac tgcgttcgaa 1020accgcgccgc agtttgaagg
taagatcggc gagcaggagt acttcgataa aggcgtgctg 1080atgatcgcga
tggtgaaagc gggcgttgag ctggcgttcg aaaccatggt cgattccggc
1140atcatcgaag aatccgctta ctacgaatca ctgcacgagc tgccgctgat
cgcgaacacc 1200atcgcccgta agcgtctgta cgaaatgaac gtggttatct
ccgataccgc agaatacggt 1260aactatctgt tctcttacgc ttgcgtaccg
ctgctgaaac cgtttattgc ggaattgcaa 1320ccgggcgatc tgggtagtgc
tatcccggaa ggcgcggtag acaacgcaca gcttcgcgac 1380gtgaacgacg
cgattcgtag tcatgcgatt gagcaggtag gtaagaaact gcgcggctat
1440atgacggata tgaagcgtat tgcggtagcg ggttga 147664491PRTSalmonella
enterica 64Met Ala Asn Tyr Phe Asn Thr Leu Asn Leu Arg Gln Gln Leu
Ala Gln 1 5 10 15 Leu Gly Lys Cys Arg Phe Met Gly Arg Asp Glu Phe
Ala Asp Gly Ala 20 25 30 Ser Tyr Leu Gln Gly Lys Lys Val Val Ile
Val Gly Cys Gly Ala Gln 35 40 45 Gly Leu Asn Gln Gly Leu Asn Met
Arg Asp Ser Gly Leu Asp Ile Ser 50 55 60 Tyr Ala Leu Arg Lys Glu
Ala Ile Ala Glu Lys Arg Ala Ser Trp Arg 65 70 75 80 Lys Ala Thr Glu
Asn Gly Phe Lys Val Gly Thr Tyr Glu Glu Leu Ile 85 90 95 Pro Gln
Ala Asp Leu Val Val Asn Leu Thr Pro Asp Lys Gln His Ser 100 105 110
Asp Val Val Arg Ser Val Gln Pro Leu Met Lys Asp Gly Ala Ala Leu 115
120 125 Gly Tyr Ser His Gly Phe Asn Ile Val Glu Val Gly Glu Gln Ile
Arg 130 135 140 Lys Asp Ile Thr Val Val Met Val Ala Pro Lys Cys Pro
Gly Thr Glu 145 150 155 160 Val Arg Glu Glu Tyr Lys Arg Gly Phe Gly
Val Pro Thr Leu Ile Ala 165 170 175 Val His Pro Glu Asn Asp Pro Lys
Gly Glu Gly Met Ala Ile Ala Lys 180 185 190 Ala Trp Ala Ala Ala Thr
Gly Gly His Arg Ala Gly Val Leu Glu Ser 195 200 205 Ser Phe Val Ala
Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile 210 215 220 Leu Cys
Gly Met Leu Gln Ala Gly Ser Leu Leu Cys Phe Asp Lys Leu 225 230 235
240 Val Ala Glu Gly Thr Asp Pro Ala Tyr Ala Glu Lys Leu Ile Gln Phe
245 250 255 Gly Trp Glu Thr Ile Thr Glu Ala Leu Lys Gln Gly Gly Ile
Thr Leu 260 265 270 Met Met Asp Arg Leu Ser Asn Pro Ala Lys Leu Arg
Ala Tyr Ala Leu 275 280 285 Ser Glu Gln Leu Lys Glu Ile Met Ala Pro
Leu Phe Gln Lys His Met 290 295 300 Asp Asp Ile Ile Ser Gly Glu Phe
Ser Ser Gly Met Met Ala Asp Trp 305 310 315 320 Ala Asn Asp Asp Lys
Lys Leu Leu Thr Trp Arg Glu Glu Thr Gly Lys 325 330 335 Thr Ala Phe
Glu Thr Ala Pro Gln Phe Glu Gly Lys Ile Gly Glu Gln 340 345 350 Glu
Tyr Phe Asp Lys Gly Val Leu Met Ile Ala Met Val Lys Ala Gly 355 360
365 Val Glu Leu Ala Phe Glu Thr Met Val Asp Ser Gly Ile Ile Glu Glu
370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His Glu Leu Pro Leu Ile Ala
Asn Thr 385 390 395 400 Ile Ala Arg Lys Arg Leu Tyr Glu Met Asn Val
Val Ile Ser Asp Thr 405 410 415 Ala Glu Tyr Gly Asn Tyr Leu Phe Ser
Tyr Ala Cys Val Pro Leu Leu 420 425 430 Lys Pro Phe Ile Ala Glu Leu
Gln Pro Gly Asp Leu Gly Ser Ala Ile 435 440 445 Pro Glu Gly Ala Val
Asp Asn Ala Gln Leu Arg Asp Val Asn Asp Ala 450 455 460 Ile Arg Ser
His Ala Ile Glu Gln Val Gly Lys Lys Leu Arg Gly Tyr 465 470 475 480
Met Thr Asp Met Lys Arg Ile Ala Val Ala Gly 485 490
657PRTArtificial SequenceAcetolactate synthase motif 65Ser Gly Pro
Gly Xaa Xaa Asn 1 5 666PRTArtificial SequenceAcetolactate synthase
motif 66Gly Xaa Xaa Gly Xaa Xaa 1 5 6715PRTArtificial
SequenceAcetolactate synthase motif 67Gly Xaa Xaa Xaa Xaa Gly Xaa
Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa 1 5 10 15 685PRTArtificial
SequenceAcetolactate synthase motif 68Gly Asp Xaa Xaa Phe 1 5
699PRTArtificial SequenceDihydroxy acid dehydratase motif 69Ser Leu
Xaa Ser Arg Xaa Xaa Ile Ala 1 5 707PRTArtificial SequenceDihydroxy
acid dehydratase motif 70Cys Asp Lys Xaa Xaa Pro Gly 1 5
7110PRTArtificial SequenceDihydroxy acid dehydratase motif 71Gly
Xaa Cys Xaa Gly Xaa Xaa Thr Ala Asn 1 5 10 725PRTArtificial
SequenceDihydroxy acid dehydratase motif 72Gly Gly Ser Thr Asn 1 5
7311PRTArtificial SequenceDihydroxy acid dehydratase motif 73Gly
Pro Xaa Gly Xaa Pro Gly Met Arg Xaa Glu 1 5 10 7410PRTArtificial
SequenceDihydroxy acid dehydratase motif 74Ala Leu Xaa Thr Asp Gly
Arg Xaa Ser Gly 1 5 10 757PRTArtificial SequenceDihydroxy acid
dehydratase motif 75Gly His Xaa Xaa Pro Glu Ala 1 5
767PRTArtificial Sequence2-keto-acid decarboxylase motif 76Phe Gly
Xaa Xaa Gly Xaa Xaa 1 5 779PRTArtificial Sequence2-keto-acid
decarboxylase motif 77Xaa Thr Xaa Gly Xaa Gly Xaa Xaa Xaa 1 5
789PRTArtificial Sequence2-keto-acid decarboxylase motif 78Asn Xaa
Xaa Ala Gly Xaa Xaa Ala Glu 1 5 796PRTArtificial
Sequence2-keto-acid decarboxylase motif 79Xaa Xaa Xaa Ile Xaa Gly 1
5 808PRTArtificial Sequence2-keto-acid decarboxylase motif 80Gly
Asp Gly Xaa Xaa Gln Xaa Thr 1 5 816PRTArtificial SequenceAlcohol
dehydrogenase motif 81Cys Xaa Xaa Asp Xaa His 1 5 828PRTArtificial
SequenceAlcohol dehydrogenase motif 82Gly His Glu Xaa Xaa Gly Xaa
Val 1 5 837PRTArtificial SequenceAlcohol dehydrogenase motif 83Xaa
Xaa Xaa Gly Xaa Xaa Xaa 1 5 847PRTArtificial SequenceAlcohol
dehydrogenase motif 84Cys Xaa Xaa Cys Xaa Xaa Cys 1 5
856PRTArtificial SequenceAlcohol dehydrogenase motif 85Xaa Xaa Xaa
Xaa Thr Xaa 1 5 866PRTArtificial SequenceAlcohol dehydrogenase
motif 86Gly Xaa Gly Xaa Xaa Gly 1 5 871044DNABacteroides
thetaiotaomicron 87atggcacagt tgaattttgg cggaactgta gaaaatgtag
ttatccgtga tgaatttcca 60ttggaaaaag ctcgtgaagt attgaaaaat gaaacaatcg
ctgtaatcgg ttatggcgta 120caaggtcctg gacaggctct gaaccttcgt
gataacggtt tcaatgtaat cgttggtcaa 180cgccagggaa agacatatga
caaagcggta gctgacggat gggttccggg tgaaactttg 240ttcggtattg
aagaagcttg cgaaaaaggt acgatcatta tgtgcctgtt gtctgatgca
300gcggtaatgt ctgtatggcc tactatcaag ccttacctga ctgcaggaaa
agctctttat 360ttctctcatg gttttgctat tacatggagt gatcgcacag
gtgtagttcc tcctgcagat 420atcgacgtaa tcatggttgc tcctaaaggt
tcgggtacat ccttgcgtac tatgttcctt 480gaaggtcgcg gcttgaactc
ttcttacgct atctatcagg atgcaacagg caacgctatg 540gacagaacaa
tcgcattggg tatcggtatc ggttcaggtt atttgttcga aacaactttc
600atccgcgaag ctacttccga cctgacaggc gaacgtggtt cattgatggg
agctatccag 660ggtctgttgc tggcacaata cgaagtgtta cgtgaaaacg
gtcacactcc ttccgaagca 720ttcaacgaaa ctgtagaaga gctgactcag
tcattgatgc cgttgtttgc aaagaacggt 780atggactgga tgtatgctaa
ctgctctact acagctcaac gtggtgctct cgactggatg 840ggccccttcc
acgatgctat caaaccggta gttgaaaagt tgtatcacag tgtgaagact
900ggtaacgaag cacagatttc aatcgactct aactccaaac cggattatcg
tgagaaactg 960gaagaagaac tgaaagcatt gcgcgaaagc gaaatgtggc
agactgccgt gacagttcgt 1020aaacttcgtc cggaaaataa ttaa
104488347PRTBacteroides thetaiotaomicron 88Met Ala Gln Leu Asn Phe
Gly Gly Thr Val Glu Asn Val Val Ile Arg 1 5 10 15 Asp Glu Phe Pro
Leu Glu Lys Ala Arg Glu Val Leu Lys Asn Glu Thr 20 25 30 Ile Ala
Val Ile Gly Tyr Gly Val Gln Gly Pro Gly Gln Ala Leu Asn 35 40 45
Leu Arg Asp Asn Gly Phe Asn Val Ile Val Gly Gln Arg Gln Gly Lys 50
55 60 Thr Tyr Asp Lys Ala Val Ala Asp Gly Trp Val Pro Gly Glu Thr
Leu 65 70 75 80 Phe Gly Ile Glu Glu Ala Cys Glu Lys Gly Thr Ile Ile
Met Cys Leu 85 90 95 Leu Ser Asp Ala Ala Val Met Ser Val Trp Pro
Thr Ile Lys Pro Tyr 100 105 110 Leu Thr Ala Gly Lys Ala Leu Tyr Phe
Ser His Gly Phe Ala Ile Thr 115 120 125 Trp Ser Asp Arg Thr Gly Val
Val Pro Pro Ala Asp Ile Asp Val Ile 130 135 140 Met Val Ala Pro Lys
Gly Ser Gly Thr Ser Leu Arg Thr Met Phe Leu 145 150 155 160 Glu Gly
Arg Gly Leu Asn Ser Ser Tyr Ala Ile Tyr Gln Asp Ala Thr 165 170 175
Gly Asn Ala Met Asp Arg Thr Ile Ala Leu Gly Ile Gly Ile Gly Ser 180
185 190 Gly Tyr Leu Phe Glu Thr Thr Phe Ile Arg Glu Ala Thr Ser Asp
Leu 195 200 205 Thr Gly Glu Arg Gly Ser Leu Met Gly Ala Ile Gln Gly
Leu Leu Leu 210 215 220 Ala Gln Tyr Glu Val Leu Arg Glu Asn Gly His
Thr Pro Ser Glu Ala 225 230 235 240 Phe Asn Glu Thr Val Glu Glu Leu
Thr Gln Ser Leu Met Pro Leu Phe 245 250 255 Ala Lys Asn Gly Met Asp
Trp Met Tyr Ala Asn Cys Ser Thr Thr Ala 260 265 270 Gln Arg Gly Ala
Leu Asp Trp Met Gly Pro Phe His Asp Ala Ile Lys 275
280 285 Pro Val Val Glu Lys Leu Tyr His Ser Val Lys Thr Gly Asn Glu
Ala 290 295 300 Gln Ile Ser Ile Asp Ser Asn Ser Lys Pro Asp Tyr Arg
Glu Lys Leu 305 310 315 320 Glu Glu Glu Leu Lys Ala Leu Arg Glu Ser
Glu Met Trp Gln Thr Ala 325 330 335 Val Thr Val Arg Lys Leu Arg Pro
Glu Asn Asn 340 345 891053DNASchizosaccharomyces pombe 89atgcgtgtta
tggactttgc cggtaccaag gagaacgttt gggagcgttc tgactggcct 60cgtgaaaagc
ttgttgacta cttcaagaac gacactcttg ccatcattgg atacggatct
120caaggacatg gtcaaggttt gaacgctcgt gatcaaggtt tgaacgttat
tgtcggtgtc 180cgtaaggatg gtgcttcctg gaagcaagct attgaagacg
gttgggtccc tggtaagact 240ttgttccccg tcgaggaggc catcaagaag
ggttctatca tcatgaacct tttgtccgat 300gccgctcaaa ctgagacttg
gcccaagatt gctcccctta ttaccaaggg taagactttg 360tacttctctc
acggtttctc cgtcatcttc aaggatcaaa ctaagattca cccccctaag
420gatgttgatg ttatccttgt cgctcccaag ggttctggtc gtaccgttcg
tacccttttc 480aaggaaggtc gtggtattaa ctcttccttc gctgtttacc
aagacgttac tggtaaggct 540caagaaaagg ccattggttt ggctgttgcc
gtcggttccg gtttcatcta ccaaaccact 600ttcaagaagg aggttatctc
cgatttggtt ggtgagcgtg gatgtctcat gggtggtatc 660aacggtcttt
tcttggctca ataccaagtt ttgcgtgaac gtggtcactc ccctgctgag
720gctttcaacg agactgttga agaggccact caatcccttt accccttgat
tggcaagtac 780ggtcttgact acatgtttgc cgcttgctct accaccgctc
gtcgtggtgc cattgactgg 840actcctcgtt tccttgaggc taacaagaag
gtccttaatg aattgtatga caatgttgag 900aacggtaacg aggctaagcg
ttccttggaa tacaactctg ctcccaacta ccgtgagctt 960tacgataagg
agttggagga aatccgcaac ttggaaatct ggaaggctgg tgaggttgtt
1020cgttctctcc gtcctgaaca caacaagcac tag
105390350PRTSchizosaccharomyces pombe 90Met Arg Val Met Asp Phe Ala
Gly Thr Lys Glu Asn Val Trp Glu Arg 1 5 10 15 Ser Asp Trp Pro Arg
Glu Lys Leu Val Asp Tyr Phe Lys Asn Asp Thr 20 25 30 Leu Ala Ile
Ile Gly Tyr Gly Ser Gln Gly His Gly Gln Gly Leu Asn 35 40 45 Ala
Arg Asp Gln Gly Leu Asn Val Ile Val Gly Val Arg Lys Asp Gly 50 55
60 Ala Ser Trp Lys Gln Ala Ile Glu Asp Gly Trp Val Pro Gly Lys Thr
65 70 75 80 Leu Phe Pro Val Glu Glu Ala Ile Lys Lys Gly Ser Ile Ile
Met Asn 85 90 95 Leu Leu Ser Asp Ala Ala Gln Thr Glu Thr Trp Pro
Lys Ile Ala Pro 100 105 110 Leu Ile Thr Lys Gly Lys Thr Leu Tyr Phe
Ser His Gly Phe Ser Val 115 120 125 Ile Phe Lys Asp Gln Thr Lys Ile
His Pro Pro Lys Asp Val Asp Val 130 135 140 Ile Leu Val Ala Pro Lys
Gly Ser Gly Arg Thr Val Arg Thr Leu Phe 145 150 155 160 Lys Glu Gly
Arg Gly Ile Asn Ser Ser Phe Ala Val Tyr Gln Asp Val 165 170 175 Thr
Gly Lys Ala Gln Glu Lys Ala Ile Gly Leu Ala Val Ala Val Gly 180 185
190 Ser Gly Phe Ile Tyr Gln Thr Thr Phe Lys Lys Glu Val Ile Ser Asp
195 200 205 Leu Val Gly Glu Arg Gly Cys Leu Met Gly Gly Ile Asn Gly
Leu Phe 210 215 220 Leu Ala Gln Tyr Gln Val Leu Arg Glu Arg Gly His
Ser Pro Ala Glu 225 230 235 240 Ala Phe Asn Glu Thr Val Glu Glu Ala
Thr Gln Ser Leu Tyr Pro Leu 245 250 255 Ile Gly Lys Tyr Gly Leu Asp
Tyr Met Phe Ala Ala Cys Ser Thr Thr 260 265 270 Ala Arg Arg Gly Ala
Ile Asp Trp Thr Pro Arg Phe Leu Glu Ala Asn 275 280 285 Lys Lys Val
Leu Asn Glu Leu Tyr Asp Asn Val Glu Asn Gly Asn Glu 290 295 300 Ala
Lys Arg Ser Leu Glu Tyr Asn Ser Ala Pro Asn Tyr Arg Glu Leu 305 310
315 320 Tyr Asp Lys Glu Leu Glu Glu Ile Arg Asn Leu Glu Ile Trp Lys
Ala 325 330 335 Gly Glu Val Val Arg Ser Leu Arg Pro Glu His Asn Lys
His 340 345 350 9120DNAArtificial SequenceT7_for primer
91taatacgact cactataggg 209219DNAArtificial SequenceT7_rev primer
92gctagttatt gctcagcgg 199340DNAArtificial
SequenceLlKARI_Y26NNK_for primer 93atcgccgtta ttggannkgg ttcacaagga
catgcccatg 409440DNAArtificial SequenceLlKARI_Y26NNK_rev primer
94catgggcatg tccttgtgaa ccmnntccaa taacggcgat 409540DNAArtificial
SequenceLlKARI_V48NNK_for primer 95caatgttatc attggtnnka ggcacggaaa
atcttttgat 409640DNAArtificial SequenceLlKARI_V48NNK_rev primer
96atcaaaagat tttccgtgcc tmnnaccaat gataacattg 409734DNAArtificial
SequenceLlKARI_R49NNK_for primer 97gttatcattg gtgtannkca cggaaaatct
tttg 349834DNAArtificial SequenceLlKARI_R49NNK_rev primer
98caaaagattt tccgtgmnnt acaccaatga taac 349939DNAArtificial
SequenceLlKARI_G51NNK_for primer 99attggtgtaa ggcacnnkaa atcttttgat
aaagctaag 3910039DNAArtificial SequenceLlKARI_G51NNK_rev primer
100cttagcttta tcaaaagatt tmnngtgcct tacaccaat 3910139DNAArtificial
SequenceLlKARI_K52NNK_for 101ggtgtaaggc acggannktc ttttgataaa
gctaaggaa 3910239DNAArtificial SequenceLlKARI_K52NNK_rev primer
102ttccttagct ttatcaaaag amnntccgtg ccttacacc 3910337DNAArtificial
SequenceLlKARI_S53NNK_for primer 103gtgtaaggca cggaaaannk
tttgataaag ctaagga 3710437DNAArtificial SequenceLlKARI_S53NNK_rev
primer 104tccttagctt tatcaaamnn ttttccgtgc cttacac
3710537DNAArtificial SequenceLlKARI_L85NNK_for primer 105tttggcacca
gatgagnnkc aacaatccat atacgag 3710637DNAArtificial
SequenceLlKARI_L85NNK_rev primer 106ctcgtatatg gattgttgmn
nctcatctgg tgccaaa 3710739DNAArtificial SequenceLlKARI_I89NNK_for
primer 107gagttgcaac aatccnnkta cgaggaggat atcaagcct
3910839DNAArtificial SequenceLlKARI_I89NNK_rev primer 108aggcttgata
tcctcctcgt amnnggattg ttgcaactc 3910958DNAArtificial
SequenceLl_recomb_1a_for primer 109gggcacaatg ttatcattgg tsyacbacac
ggamwatctt ttgataaagc taaggaag 5811058DNAArtificial
SequenceLl_recomb_1b_for primer 110gggcacaatg ttatcattgg tsyagtgcac
ggamwatctt ttgataaagc taaggaag 5811158DNAArtificial
SequenceLl_recomb_1c_for primer 111gggcacaatg ttatcattgg tsyatcgcac
ggamwatctt ttgataaagc taaggaag 5811258DNAArtificial
SequenceLl_recomb_1a_rev primer 112cttccttagc tttatcaaaa gatwktccgt
gtvgtrsacc aatgataaca ttgtgccc 5811358DNAArtificial
SequenceLl_recomb_1b_rev primer 113cttccttagc tttatcaaaa gatwktccgt
gcactrsacc aatgataaca ttgtgccc 5811458DNAArtificial
SequenceLl_recomb_1c_rev primer 114cttccttagc tttatcaaaa gatwktccgt
gcgatrsacc aatgataaca ttgtgccc 5811548DNAArtificial
SequenceLl_recomb_2a_for primer 115ggcaccagat gagrcacaac aatccatata
cgaggaggat atcaagcc 4811648DNAArtificial SequenceLl_recomb_2b_for
primer 116ggcaccagat gagrcacaac aatccgcata cgaggaggat atcaagcc
4811748DNAArtificial SequenceLl_recomb_2c_for primer 117ggcaccagat
gagttgcaac aatccatata cgaggaggat atcaagcc 4811848DNAArtificial
SequenceLl_recomb_2d_for primer 118ggcaccagat gagttgcaac aatccgcata
cgaggaggat atcaagcc 4811948DNAArtificial SequenceLl_recomb_2a_rev
primer 119ggcttgatat cctcctcgta tatggattgt tgtgyctcat ctggtgcc
4812048DNAArtificial SequenceLl_recomb_2b_rev primer 120ggcttgatat
cctcctcgta tgcggattgt tgtgyctcat ctggtgcc 4812148DNAArtificial
SequenceLl_recomb_2c_rev primer 121ggcttgatat cctcctcgta tatggattgt
tgcaactcat ctggtgcc 4812248DNAArtificial SequenceLl_recomb_2d_rev
primer 122ggcttgatat cctcctcgta tgcggattgt tgcaactcat ctggtgcc
4812330DNAArtificial SequenceLl_recomb_3KS_for primer 123cacggaaaat
cttttgataa agctaaggaa 3012430DNAArtificial
SequenceLl_recomb_3LS_for primer 124cacggactat cttttgataa
agctaaggaa 3012530DNAArtificial SequenceLl_recomb_3KD_for primer
125cacggaaaag attttgataa agctaaggaa 3012630DNAArtificial
SequenceLl_recomb_3LD_for primer 126cacggactag attttgataa
agctaaggaa 3012730DNAArtificial SequenceLl_recomb_3KS_rev primer
127ttccttagct ttatcaaaag attttccgtg 3012830DNAArtificial
SequenceLl_recomb_3LS_rev primer 128ttccttagct ttatcaaaag
atagtccgtg 3012930DNAArtificial SequenceLl_recomb_3KD_rev primer
129ttccttagct ttatcaaaat cttttccgtg 3013030DNAArtificial
SequenceLl_recomb_3LD_rev primer 130ttccttagct ttatcaaaat
ctagtccgtg 3013136DNAArtificial SequenceLl_K52NNkS53NNK_for primer
131ggtctaccac acggannknn ktttgataaa gctaag 3613236DNAArtificial
SequenceLl_K52NNkS53NNK_rev primer 132cttagcttta tcaaamnnmn
ntccgtgtgg tagacc 3613333DNAArtificial SequenceE59K_recomb_rev
primer 133aaaagtttcg aatccatctt ycttagcttt atc 3313433DNAArtificial
SequenceE59K_recomb_for primer 134gataaagcta agraagatgg attcgaaact
ttt 3313533DNAArtificial SequenceA70V_recomb_rev primer
135atctgcctta gctactrctt cacctacttc aaa 3313633DNAArtificial
SequenceA70V_recomb_for primer 136tttgaagtag gtgaagyagt agctaaggca
gat 3313739DNAArtificial SequenceK118E/D122G_recomb_for primer
137ggatacatcr aagtcccaga ggrcgtggac gtgtttatg 3913839DNAArtificial
SequenceK118E/D122G_recomb_rev primer 138cataaacacg tccacgycct
ctgggactty gatgtatcc 3913933DNAArtificial SequenceH135L _recomb_rev
primer 139ggtccttcta acaaggwggc ctggtgcttt tgg 3314033DNAArtificial
SequenceH135L_recomb_for primer 140ccaaaagcac caggccwcct tgttagaagg
acc 3314133DNAArtificial SequenceT182S_recomb_rev primer
141ctcttccttg aaagtgsttt caatgatgcc gac 3314233DNAArtificial
SequenceT182S_recomb_for primer 142gtcggcatca ttgaaascac tttcaaggaa
gag 3314333DNAArtificial SequenceE320K_recomb_rev primer
143catagcttgt ctaagttytg cccctatctt ttc 3314433DNAArtificial
SequenceE320K _recomb_for primer 144gaaaagatag gggcaraact
tagacaagct atg 3314532DNAArtificial SequenceSh_S78D_for primer
145gcacaaaaga gagccgattg gcaaaaagcg ac 3214632DNAArtificial
SequenceSh_ S78D_rev primer 146gtcgcttttt gccaatcggc tctcttttgt gc
3214735DNAArtificial SequenceSe2_S78D_for primer 147gcagaaaaga
gagccgattg gcgtaaagcg acgga 3514835DNAArtificial
SequenceSe2_S78D_rev primer 148tccgtcgctt tacgccaatc ggctctcttt
tctgc 35
* * * * *